US010722444B2

(12 ) United States Patent ( 10 ) Patent No.: US 10,722,444 B2 Gousse et al. (45 ) Date of Patent: Jul. 28 , 2020

(54 ) STABLE HYDROGEL COMPOSITIONS 4,605,691 A 8/1986 Balazs et al . 4,636,524 A 1/1987 Balazs et al. INCLUDING ADDITIVES 4,642,117 A 2/1987 Nguyen et al. 4,657,553 A 4/1987 Taylor (71 ) Applicant: Allergan Industrie, SAS , Pringy (FR ) 4,713,448 A 12/1987 Balazs et al . 4,716,154 A 12/1987 Malson et al. ( 72 ) 4,772,419 A 9/1988 Malson et al. Inventors: Cécile Gousse , Dingy St. Clair ( FR ) ; 4,803,075 A 2/1989 Wallace et al . Sébastien Pierre, Annecy ( FR ) ; Pierre 4,886,787 A 12/1989 De Belder et al . F. Lebreton , Annecy ( FR ) 4,896,787 A 1/1990 Delamour et al. 5,009,013 A 4/1991 Wiklund ( 73 ) Assignee : Allergan Industrie , SAS , Pringy (FR ) 5,087,446 A 2/1992 Suzuki et al. 5,091,171 A 2/1992 Yu et al. 5,143,724 A 9/1992 Leshchiner ( * ) Notice : Subject to any disclaimer , the term of this 5,246,698 A 9/1993 Leshchiner et al . patent is extended or adjusted under 35 5,314,874 A 5/1994 Miyata et al. U.S.C. 154 (b ) by 0 days. 5,328,955 A 7/1994 Rhee et al . 5,356,883 A 10/1994 Kuo et al . (21 ) Appl. No.: 15 /514,329 5,399,351 A 3/1995 Leshchiner et al . 5,428,024 A 6/1995 Chu et al . 5,531,716 A 7/1996 Luzio et al . ( 22 ) PCT Filed : Sep. 30 , 2014 5,565,519 A 10/1996 Rhee et al . 5,571,503 A 11/1996 Mausner (86 ) PCT No .: PCT / IB2014 /001959 5,614,587 A 3/1997 Rhee et al. 5,616,568 A 4/1997 Pouyani et al. § 371 ( c ) ( 1 ) , 5,616,611 A 4/1997 Yamamoto et al . (2 ) Date: Mar. 24 , 2017 5,616,689 A 4/1997 Shenoy et al . 5,633,001 A 5/1997 Agerup ( 87 ) PCT Pub . No .: WO2016 /051219 5,643,464 A 7/1997 Rhee et al . 5,676,964 A 10/1997 Della Valle et al . PCT Pub . Date : Apr. 7 , 2016 (Continued ) (65 ) Prior Publication Data FOREIGN PATENT DOCUMENTS US 2017/0273886 A1 Sep. 28 , 2017 CA 949965 6/1974 (51 ) Int. Ci. CA 2805008 1/2012 A61K 8/73 (2006.01 ) (Continued ) A61L 27/20 ( 2006.01) A61L 27/50 (2006.01 ) OTHER PUBLICATIONS A61K 8/04 ( 2006.01 ) A61K 8/365 ( 2006.01 ) Kim et al ., " Gallotannin Isolated from Euphorbia Species, 1,2,6 A61Q 19/08 ( 2006.01 ) Tri- O -galloyl - b - D - allose , Decreases Nitric ... " , Jun . 2009 , Bio A61K 8/49 ( 2006.01 ) logical and Pharmaceutical Bulletin , vol. 32 , issue 6 , pp . 1053-1056 . (Year : 2009 ). * ( 52 ) U.S. CI. National Center for Biotechnology Information . PubChem Com CPC A61K 8/735 (2013.01 ) ; A61K 8/042 pound Database ; CID = 370 , https://pubchem.ncbi.nlm.nih.gov/ (2013.01 ) ; A61K 8/365 ( 2013.01 ) ; A61K 8/498 compound /370 ( accessed Mar. 18 , 2018) . (Year : 2018 ). * (2013.01 ) ; A61L 27/20 ( 2013.01) ; A61L 27/50 National Center for Biotechnology Information . PubChem Com (2013.01 ) ; A61Q 19/08 (2013.01 ) ; ACIK pound Database ; CID = 689043, https://pubchem.ncbi.nlm.nih.gov/ 2800/91 (2013.01 ); A6IL 2400/06 (2013.01 ) compound /689043 (accessed Mar. 18 , 2018 ) . ( Year: 2018 ). * (58 ) Field of Classification Search Nadim et al. , “ Improvement of polyphenol properties upon glucosylation ??? A61K 2800/91 ; A61K 8/042 ; A61K 8/365 ; in a UV- induced skin cell ageing model” , first published Sep. 5 , A61K 8/498 ; A61K 8/735 ; A61Q 19/08 2014 , International Journal of Cosmetic Science , vol. 36 , pp . See application file for complete search history. 579-587 . ( Year : 2014 ). * ( Continued ) (56 ) References Cited U.S. PATENT DOCUMENTS Primary Examiner Michael B. Pallay 2,128,827 A 8/1938 Killian (74 ) Attorney, Agent, or Firm — Nathan S. Smith ; Chris 3,548,056 A 12/1970 Eigen et al. Betti ; Morgan , Lewis & Bockius LLP 3,763,009 A 10/1973 Suzuki et al. 3,949,073 A 4/1976 Daniels et al. 4,060,081 A 11/1977 Yannas et al . (57 ) ABSTRACT 4,140,537 A 2/1979 Luck et al . 4,233,360 A 11/1980 Luck et al. The present specification generally relates to an injectable 4,273,705 A 6/1981 Kato 4,279,812 A 7/1981 Cioca dermal filler composition that includes crosslinked 4,424,208 A 1/1984 Wallace et al . hyaluronic acid -based polymer and an antioxidant . 4,501,306 A 2/1985 Chu et al . 4,582,640 A 4/1986 Smestad et al. 4,582,865 A 4/1986 Balazs et al. 14 Claims, 6 Drawing Sheets US 10,722,444 B2 Page 2

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Lamar et al. , “ Antifibrosis Effect of Novel Gels in Anterior Ciliary Frati et al. , “ Degradation of Hyaluronic Acid by Photosensitized Sclerotomy (ACS ), ” The Association for Research in Vision and Riboflavin In Vitro . Modulation of the Effect by Transition Metals , Ophthalmology, Inc., 2002, 1 page . Radical Quenchers , and Metal Chelators ,” Free Radical Biology Levy et al ., “ Lidocaine Hypersensitivity After Subconjunctival Medicine , 1996 , vol. 22 , No. 7 , pp . 1139-1144 . Injection , ” Can J Ophthalmol , 2006 , vol. 41, 204-206 . Fujinaga et al. , “ Reproductive and Teratogenic Effects of Lidocaine Lindvall et al ., “ Influence of Various Compounds on the Degrada in Sprague- Dawley Rats ,” Anesthesiology, 1986 , vol . 65 , pp . 626 tion of Hyaluronic Acid by a Myeloperoxidase System ,” Chemico 632 . Biological Interactions, 1994 , vol . 90 , pp . 1-12 . 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( 56 ) References Cited Wang et al. , “ Development of hyaluronic acid -based scaffolds for brain tissue engineering ,” Acta Biomaterialia, 2009 , pp . 2371-2384 . OTHER PUBLICATIONS Waraszkiewicz et al. , “ Stability -Indicating High -Performance Liq uid Chromatographic Analysis of Lidocaine Hydrochloride and Sannino et al ., " Crosslinking of Cellulose Derivatives and Hyaluronic Acid With Water - Soluble Carbodiimide, ” Polymer, 2005 , vol. 46 , Lidocaine Hydrochloride with Epinephrine Injectable Solutions, " pp . 11206-11212 . Journal of Pharmaceutical Sciences, 1981 , vol. 70 , No. 11, pp . Sculptra Product Information , Dermik Laboratories, Jun . 2004 , 12 1215-1218 . pages . Weidmann , " New Hyaluronic Acid Filler for Subdermal and Long Segura et al. , “ Crosslinked Hyaluronic Acid Hydrogels : A Strategy lasting Volume Restoration of the Face, ” European Dermatology , to Functionalize and Pattern ,” Biomaterials, 2005 , vol. 26 , No. 4 , 2009 , pp . 65-68 . pp . 359-371. Xia et al. , “ Comparison of Effects of Lidocaine Hydrochloride , Selvi et al ., “ Arthritis Induced by Corticosteroid Crystals, ” The Buffered Lidocaine, Diphenhydramine, and Normal Saline After Journal of Rheumatology, 2004, vol. 31 , No. 3 , pp . 622 . Intradermal Injection ,” Journal of Clinical Anesthesia, 2002 , vol. Shu et al, “ Synthesis and evaluation of injectable , in situ crosslink 14 , pp . 339-343 . able synthetic extracellular matrices for tissue engineering , ” Journal Yeom et al. , “ Effect of Cross- Linking Reagents for Hyaluronic Acid of Biomedical Materials Research , 2006 , vol. 79A , pp . 902-912 . Hydrogel Dermal Fillers on Tissue Augmentation and Regenera Silver et al. , “ Physical Properties of Hyaluronic Acid and tion ,” Bioconjugate Chemistry , 2010 , vol. 21, pp . 240-247 . Hydroxypropylmethylcellulose in Solution : Evaluation of Coating Yui et al. , “ Inflammation Responsive Degradation of Crosslinked Ability ,” Journal of Applied Biomaterials, 1994 , vol. 5 , pp . 89-98 . Hyaluronic Acid Gels, ” Journal of Controlled Release , 1992 , vol. Skardal et al. , “ Bioprinting Vessel -Like Constructs Using Hyaluronan 26 , pp . 105-116 . Hydrogels Crosslinked With Tetrahedral Polyethylene Glycol Yui et al. , “ Photo -Responsive Degradation of Heterogeneous Hydrogels Tetracrylates, ” Biomaterials , 2010 , vol. 31, pp . 6173-6181. Comprising Crosslinked Hyaluronic Acid and Lipid Microspheres Smith et al. , “ Five Percent Lidocaine Cream Applied Simultane for Temporal Drug Delivery, ” Journal of Controlled Release, 1993, ously to the Skin and Mucosa of the Lips Creates Excellent vol . 26 , pp . 141-145 . Anesthesia for Filler Injections, ” Dermatol Surg , 2005, vol. 31, pp . Yun et al. , “ Hyaluronan Microspheres for Sustained Gene Delivery 1635-1637 . and Site - Specific Targeting, ” Biomaterials , 2004 , vol. 25 , pp . 147 Tezel et al. , “ The science ofhyaluronic acid dermal fillers ,” Journal 157 . of Cosmetic and Laser Therapy , 2008 , vol. 10 , pp . 35-42 . Zheng et al ., “ In situ crosslinkable hyaluronan hydrogels for tissue Tomihata et al. , “ Crosslinking of Hyaluronic Acid with Water engineering, ” Biomaterials , 2004 , vol. 25 , pp . 1339-1348 . Soluable Carbodiimide ,” J Biomed Mater Res , Feb. 1997 , vol . 37 , Zulian et al. , “ Triamcinolone acetonide and hexacetonide intra No. 2 , pp . 243-251 . articular treatment of symmetrical joints in juvenile idiopathic Visiol, TRB Chemedica Ophthalmic Line, Product Info , May 2014 , arthritis: a double -blind trial, ” Rheumatology , Oct. 2004 , vol. 43 , p . 1-2 , Geneva , CH . No. 10 , pp . 1288-1291 . Visiol , Viscoelstic Gel for Use in Ocular Surgery , http : //www . Lim et al ., “ Regioselectivity of glucosylation of caffeic acid by a trbchemedica.com/index.php/option=com_content&tas , 2010, 1 Page. UDP - glycose : glucosyltransferase is maintained in planta ,” Biochem Wahl, “ European Evaluation of a New Hyaluronic Acid Filler J. 2003 , vol . 373 , No. 3 , pp . 987-992 . Incorporating Lidocaine, ” Journal of Cosmetic Dermatology , 2008 , vol. 7 , pp . 298-303 . * cited by examiner U.S. Patent Jul. 28 ,9 2020 Sheet 1 of 6 US 10,722,444 B2

350 300 250 205 1HzPa'()G 150 100 50

-10 0 10 20 30 40 50 60 Time in days at 45 ° C

• control Inoveol CAFA

FIG . 1

U.S. Patent Jul. 28 ,9 2020 Sheet 3 of 6 US 10,722,444 B2

25

20 y = -0.0028x + 23.789 15 Pa.s kokelaatat 10 y = -0,0041X + 23,3 5

0 500 1000 1500 2000 2500 3000 3500 4000 time (s )

..X** • control CAFA

FIG . 4

25

20

15 Pa.s y = -0.0010x + 14,5688 10

5 y = -0.0023x + 16.7098

0 500 1000 1500 2000 2500 3000 3500 4000 time (s )

...Kr . control otor . CAFA

FIG 5 U.S. Patent Jul. 28 ,9 2020 Sheet 4 of 6 US 10,722,444 B2

400 350 300

G'(Pa)1Hz 200 150 100 50 o -10 0 10 20 30 40 50 60 Time in days at 45 ° C

Control 0,5 % Inoveol EGCG 0,5 %

Fig 6

70 60 50 40 G"(Pa)1Hz 30

20 10

o - 10 0 10 20 30 40 50 60 Time in days at 45 ° C

• Control 0,5 % Inoveol EGCG 0,5 %

FIG . 7 U.S. Patent Jul. 28 ,9 2020 Sheet 5 of 6 US 10,722,444 B2

0.35 0.3 0.25 , 0.2

deltaTan 0.15

0.1 0.05

-10 10 20 30 40 50 60 Time in days at 45 ° C

..... Control 0,5 % Inoveol EGCG 0,5 %

FIG . 8

35 y = -0.0019x + 30.6941 30 25 20 15 10 y = -0.0039x + 25.982 5

0 500 1000 1500 2000 2500 3000 3500 4000 time (s ) . *** . control Inoveol EGCG

FIG 9 U.S. Patent Jul. 28 ,9 2020 Sheet 6 of 6 US 10,722,444 B2

35 30 y = -0.0006x + 22.0077 25 20 Pa.s 15 10 5 y = -0.0022x + 17.163

0 500 1000 1500 2000 2500 3000 3500 4000 time (s ) control Inoveol EGCG

FIG . 10 US 10,722,444 B2 1 2 STABLE HYDROGEL COMPOSITIONS hyaluronidase and chemical degradation via free radicals . INCLUDING ADDITIVES Thus, while still in commercial use , compositions compris ing uncrosslinked hyaluronan polymers tend to degrade CROSS REFERENCE TO RELATED within a few days after administration and thus require fairly APPLICATIONS 5 frequent reinjection to maintain their skin improving effect . To minimize the effect of these in vivo degradation This application is a national phase application under 35 pathways, matrix polymers are crosslinked to one another to U.S.C. § 371 of PCT Application Serial No. PCT/ IB2014 / form a stabilized hydrogel. Because hydrogels comprising 001959 , filed Sep. 30 , 2014 , the entire disclosure of this crosslinked matrix polymers are a more solid substance , application being incorporated herein by this specific refer- 10 dermal fillers comprising such hydrogels remain in place at ence . the implant site longer . In addition , these hydrogels are more suitable as a dermal filler because it is more solid nature BACKGROUND improves the mechanical properties of the filler , allowing the filler to better lift and fill a skin region . Hyaluronan poly Skin aging is a progressive phenomenon that occurs over 15 mers are typically crosslinked with a crosslinking agent to time and can be affected by lifestyle factors , such as alcohol form covalent bonds between hyaluronan polymers. Such consumption , tobacco , and sun exposure . Aging of the facial crosslinked polymers form a less water soluble hydrogel skin can be characterized by atrophy, slackening , and fat network that is more resistant to degradation , and thus tening . Atrophy corresponds to a massive reduction of the requires less frequent reinjection , than the non -crosslinked thickness of skin tissue. Slackening of the subcutaneous 20 hyaluronan compositions . tissues leads to an excess of skin and ptosis and leads to the Current dermal fillers can be associated with a variety of appearance of drooping cheeks and eyelids. Fattening refers side effects . For example , administration of a dermal filler to to an increase in excess weight by swelling of the bottom of an individual is typically performed using a syringe or the face and neck . These changes are typically associated needle . Such administration could result in one or more with dryness, loss of elasticity , and rough texture . 25 unwanted side- effects , such as, e.g., pain and discomfort to Hyaluronan , also known as hyaluronic acid (“ HA " ) , is a the individual, bleeding in and under the site of administra non - sulfated glycosaminoglycan that is distributed widely tion , and itching, inflammation and irritation in the vicinity throughout the human body in connective , epithelial, and of the administration site during and after the administration neural tissues. Hyaluronan is abundant in the different layers of the dermal filler. The dermal fillers disclosed in the of the skin , where it has multiple functions such as, e.g., to 30 present specification address these and other unwanted side ensure good hydration , to assist in the organization of the effects by providing hydrogel compositions comprising extracellular matrix , to act as a filler material ; and to agents that reduce, step , or prevent one or more of these participate in tissue repair mechanisms. Ho er, with age, side - effects . the quantity of hyaluronan , collagen , elastin , and other Additionally, a dermal filler formulation must be capable matrix polymers present in the skin decreases. For example , 35 of withstanding sterilization which is a strict requirement repeated exposed to ultra violet light , e.g., from the sun , before the product can be sold (the product must be sterile ) . causes dermal cells to both decrease their production of Sterilization can be carried out by steam sterilization , filtra hyaluronannan as well as increase the rate of its degradation . tion , microfiltration , gamma radiation , ETO light or by a This hyaluronan loss results in various skin conditions such combination of these methods . It is known that a dermal as , e.g., imperfects , defects , diseases and/ or disorders , and 40 filler can be steam sterilized ( autoclaved ) without substantial the like . For instance, there is a strong correlation between degradation of physical properties, but when a dermal filler the water content in the skin and levels of hyaluronan in the formulation contains an additional labile ingredient ( such as dermal tissue . As skin ages, the amount and quality of an antioxidant, anti - itch agent, an anti -cellulite agent, an hyaluronan in the skin is reduced . These changes lead to anti- scarring agent, an anti- inflammatory agent, an anes drying and wrinkling of the skin . 45 thetic agent, an anti - irritant agent, a vasoconstrictor, a vaso Dermal fillers are useful in treating soft tissue condition dilator, an anti -hemorrhagic agent like a hemostatic agent or and in other skin therapies because the fillers can replace lost anti - fibrinolytic agent, a desquamating agent, a tensioning endogenous matrix polymers, or enhance / facilitate the func agent, an anti- acne agent , a pigmentation agent, an anti tion of existing matrix polymers , in order to treat these skin pigmentation agent, or a moisturizing agent) the entire conditions. In the past, such compositions have been used in 50 dermal filler formulation or at least the additional (heat cosmetic applications to fill wrinkles , lines , folds , scars , and labile ) agent is traditionally sterilized by a non -heat treat to enhance dermal tissue, such as, e.g., to plump thin lips , or ment such as by a filtration sterilization method . Thus, a fill - in sunken eyes or shallow cheeks. One common matrix known dermal filler product (REVITACARE® Bio - Revit polymer used in dermal filler compositions is hyaluronan . alisation , REVITACARE® Laboratory, Saint -Ouen Because hyaluronan is natural to the human body, it is a 55 l’Aumône , France) is sold in two separate vials or contain generally well tolerated and a fairly low risk treatment for a ers , one vial containing the HA ( which is autoclave wide variety of skin conditions . sterilized )) and the second vial containing any additional Originally , compositions comprising hyaluronan where ingredients ( the second vial contents are sterilized by filtra made from naturally occurring polymers , which exist in an tion ). Another known dermal filler product NCTF 135HA uncrosslinked state . Although exhibiting excellent biocom- 60 (Laboratoires Filorga, Paris, France) is sold in a single patibility and affinity for water molecules, naturally occur container holding both hyaluronan and any additional ingre ring hyaluronan exhibits poor biomechanical properties as a dients , all having been sterilized by microfiltration . The dermal filler . One primary reason is that because this poly dermal fillers disclosed in the present specification addresses mer is uncrosslinked , it is highly soluble and , as such , is this issue by developing dermal fillers that are entirely cleared rapidly when administered into a skin region . This in 65 sterilized by a heat treatment, i.e. , in some embodiments of vivo clearance is primarily achieved by rapid degradation of this invention , none of the components are sterilized solely the polymers, principally enzymatic degradation via using a non -heat treatment such as, e.g. , filtration . US 10,722,444 B2 3 4 SUMMARY ing a heat treatment of at least 100 ° C. Advantageously , the heat sterilized composition is substantially stable at room The present specification provides novel dermal fillers temperature for up to at least about 3 months, for example , useful for treating skin conditions that remain stable after a at least about 12 months, at least about 24 months, or at least heat treatment used to sterilize the compositions. One aspect 5 about 36 months . of the disclosed dermal fillers , and a significant distinction In some embodiments , the antioxidant agent can be a over known dermal fillers , is that dermal fillers disclosed herein are prepared by : (1 ) mixing glycosaminoglycan poly phenolic molecule. Non - limiting examples of phenolic mol mers and the additional agents ( s ) disclosed herein , and then ; ecules suitable in embodiments of the present invention ( 2 ) heat treating the dermal filler composition to at least 100 ° 10 include ; caffeic acid , catechin , epigallocatechin gallate , C. (no filtration sterilization of any component ) ; ( 3 ) where kaempferol, quercetin , and luteolin . In some embodiments , such treatmentmaintains the desired properties of the hydro the antioxidant agent it a glucosidic derivative of a phenolic gel compositions . In some embodiments , the disclosed agent. hydrogel compositions do not exhibit any significant deg In some embodiments , the antioxidant is water - soluble , radation as shown by pre and post autoclaved testing. In 15 and is preferably the glucosylated derivative of a non some embodiments , the disclosed hydrogel compositions are water - soluble antioxidant. For example , the antioxidant one substantially heat stable as determined by the retention of of a glucosylated caffeic acid , a glucosylated epicatechin one or more of the following characteristics after steriliza gallate or a glucosylated epigallocatechin gallate . tion : clarity (transparency and translucency ) , homogeneous In some embodiments , the phenolic agent is directly ness , extrusion force , cohesiveness, hyaluronan concentra- 20 incorporated into a hyaluronic acid based gel, for example , tion , agent (s ) concentration , osmolarity , pH , or other a crosslinked hyaluronic acid based gel. In these embodi rheological characteristics desired by the hydrogel before ments, such direct incorporation of antioxidizing agents the heat treatment. increases the resistance of the gel composition to oxidative The hydrogel compositions disclosed herein can also stress such as free radicals . Typically , in a free radical exhibit greater stability than a hydrogel composition without 25 degradation test where gel compositions are exposed to the additional constituent. Additionally , the additional ingre hydrogen peroxide and the complex viscosity or elastic dient can be hydrophilic and provides protection to the modulus of the compositions are followed by oscillation glycosaminoglycan polymers from degradation during rheology, the incorporation of antioxidant agents increases steam sterilization and /or after administration of the dermal the time it takes until the visco -elastic properties drop to a filler formulation to a patient. Without wishing to be bound 30 certain level , or drop by a given percentage. Typically , T30 , by theory , the incorporation of an additional ingredient in the the time it takes to lose 70 % of the initial elastic modulus or dermal filler formulation may inhibit free -radical scaveng complex viscosity is used to rank formulations. In some ing at the injection / implant site , thereby prolonging dermal embodiments , the HA composition comprising crosslinked filler duration after patient administration . HA and the antioxidant provides at least about a 25 % Thus, aspect of the present specification provide a hydro- 35 increase for T30 , preferably at least about a 50 % increase for gel composition comprising a glycosaminoglycan polymer T30 , and even more preferably at least about a 100 % increase and one or more antioxidant agents . for T30 Some aspects of the present specification provide a In some embodiments , the free radical scavenger proper method of preparing a hydrogel composition disclosed ties of the gel obtained according to the present disclosure herein , the method comprising ( a )mixing the glycosamino- 40 showed an improvement in the stability of the formulation glycan polymer and the at least one agent; and (b ) heat when compared with a gel without the additive . treating the mixture, wherein the heat treatment maintains In some embodiments , the composition further comprises the desired hydrogel properties disclosed herein . an anesthetic agent, for example , lidocaine or a similar Some aspects of the present specification provide a agent, present in an amount of about 0.1 % ( w / w ) to about method of treating a skin condition in an individual in need 45 1.0 % (w /w ) of the total composition . In some embodiments , thereof , the method comprising the steps of administering , lidocaine is present in an amount of about 0.3 % w /w . In for example , by subdermal injection , a hydrogel composi some embodiments , the composition further comprises an tion disclosed herein into a dermal region of the individual, antioxidant agent, for example , mannitol present in an wherein the administration improves the skin condition . amount of about 0.01 % ( w / w ) to about 5 % ( w / w ) of the total Skin conditions treated by the disclosed compositions 50 composition . In some embodiments , the hyaluronic acid include, without limitation , augmentations , reconstructions , based polymer is present at a concentration of about 5 mg/ g diseases , disorders , defects , or imperfections of a body part, to about 40 mg/ g , and comprises a low molecular weight region or area . In one aspect , a skin condition treated by the hyaluronan polymer having a mean molecular weight disclosed compositions include, without limitation , a facial greater than 300,000 Da and less than about 800,000 Da, for augmentation , a facial reconstruction , a facial disease , a 55 example , a mean molecular weight greater than 2,000,000 facial disorder, a facial defect , or a facial imperfection . In Da and less than about 5,000,000 Da. In some embodiments , one aspect , a skin condition treated by the disclosed com the hyaluronic acid -based polymer comprises both high positions include , without limitation , skin dehydration , a molecular weight hyaluronan and low molecular weight lack of skin elasticity , skin roughness, a lack of skin taut hyaluronan , wherein the high molecular weight hyaluronan ness , a skin stretch line or mark , skin paleness , a dermal 60 has a molecular weight greater than 2,000,000 Da and divot, a sunken cheek , a thin lip , a retro -orbital defect, a wherein the low molecular weighthyaluronan has a molecu facial fold , or a wrinkle . lar weight of less than 1,000,000 Da . In some embodiments a hydrogel composition comprising a hyaluronic acid -based polymer and at least one antioxidant BRIEF DESCRIPTION OF DRAWINGS is provided , wherein the hydrogel composition is sterilized 65 by heat treatment and / or pressure treatment, for example , by FIG . 1 is a graph illustrating experimental data obtained autoclaving , for example , is sterilized in a process compris that demonstrates the effect of an accelerated aging/ accel US 10,722,444 B2 5 6 erated degradation at 45 ° C. for 60 days on the elastic FIG . 10 is a graph illustrating experimental data obtained modulus G ' of the same gel with and without Inoveol® that shows that the gel containing Inoveol® EGCG at 0.5 % CAFA at 0.5 % ( w / w ) . ( w / w ) is more stable with respect to free radical degradation FIG . 2 is a graph illustrating experimental data obtained (ratio 2.0 ) , than the control, even with both gels having been that demonstrates the effect of an accelerated aging at 45 ° C. 5 submitted to accelerated aging at 45 ° C. for 60 days prior to for 60 days on the viscous modulus G " of the same gel with degradation . and without Inoveol® CAFA at 0.5 % ( w / w ). FIG . 3 is a graph illustrating experimental data obtained DETAILED DESCRIPTION that demonstrates the effect of an accelerated aging at 45 ° C. for 60 days on the tan delta of the same gel with and without 10 Aspects of the present specification provide, in part , a Inoveol® CAFA at 0.5 % ( w / w ) . hydrogel composition comprising a glycosaminoglycan FIG . 4 is a graph illustrating experimental data obtained polymer . The hydrogel composition disclosed herein can that demonstrates that the degradation of a gel containing further comprise two or more different glycosaminoglycan 0.5 % ( w / w ) Inoveol® CAFA , followed by its viscosity polymers . As used herein , the term “ glycosaminoglycan ” is decrease over time in presence of hydrogen peroxide, is 15 synonymous with “GAG ” and “ mucopolysaccharide” and slower when compared to the same gel without Inoveol® refers to long unbranched polysaccharides consisting of a CAFA . repeating disaccharide units . The repeating unit consists of FIG . 5 is a graph illustrating experimental data obtained a hexose (six -carbon sugar ) or a hexuronic acid , linked to a that demonstrates showed that the gel containing Inoveol® hexosamine ( six -carbon sugar containing nitrogen ) and CAFA is still more stable with respect to free radical 20 pharmaceutically acceptable salts thereof. Members of the degradation ( ratio of 1.5 ) after 60 days of accelerated aging GAG family vary in the type of hexosamine , hexose or at 45 ° C. hexuronic acid unit they contain , such as, e.g., glucuronic FIG . 6 is a graph illustrating experimental data obtained acid , iduronic acid , galactose, galactosamine, glucosamine ) that demonstrates the effect of an accelerated aging at 45 ° C. and may also vary in the geometry of the glycosidic linkage . for 60 days on the elastic modulus G ' of the same gel with 25 Any glycosaminoglycan polymer is useful in the hydrogel and without Inoveol® EGCG at 0.5 % ( w / w ) . compositions disclosed herein with the proviso that the FIG . 7 is a graph that further illustrates the effect of an glycosaminoglycan polymer improves a condition of the accelerated aging at 45 ° C. for 60 days on the viscous skin . Non - limiting examples of glycosaminoglycans include modulus G " of the same gel with and without Inoveol® chondroitin sulfate , dermatan sulfate , keratan sulfate , EGCG at 0.5 % ( w / w ) . 30 hyaluronan . Non - limiting examples of an acceptable salt of FIG . 8 is a graph that further illustrates the effect of an a glycosaminoglycan includes sodium salts , potassium salts , accelerated aging at 45 ° C. for 60 days on the tan delta of the magnesium salts , calcium salts, and combinations thereof. same gel with and without Inoveol® EGCG at 0.5 % ( w / w ) . GAGs useful in the hydrogel compositions and methods FIG . 9 is a graph illustrating experimental data obtained disclosed herein include those commercially available , such that demonstrates effect of Inoveol- EGCG on HA -based gel 35 as, e.g., hyaluronan -based dermal fillers JUVEDERM® , matrix oxidative degradation using hydrogen peroxide. The JUVEDERM® 30 , JUVEDERM® Ultra , JUVEDERM® value of the slope characterizes the speed of free radical Ultra Plus, JUVEDERM® Ultra XC , and JUVEDERM® degradation and shows the benefit of adding Inoveol® Ultra Plus XC (Allergan Inc, Irvine , Calif .) . Table 1 lists EGCG at 0.5 % ( w / w ) . representative GAGs. TABLE 1 Examples of GAGS Glycosidic Hexuronic linkage Name acid /Hexose Hexosamine geometry Unique features Chondroitin GICUA or GalNAc or -4GLcUAB1 Most prevalent GAG sulfate GlcUA ( 2S ) GalNAc (4S ) or 3GalNAcß1 GalNAc (6S ) or GalNAc( 48,6S ) Dermatan GICUA or GalNAc or -4IdoUAB1 Distinguished from chondroitin sulfate IDOUA or GalNAc ( 4S ) or 3GalNAcß1 sulfate the presence of iduronic IdoUA ( 2S ) GalNAc( 6S ) or acid , although some hexuronic GalNAc ( 48,6S ) acid monosaccharides may be glucuronic acid . Keratan Gal or GlcNAc or -3Gal (6S )B1 Keratan sulfate type II may be sulfate Gal( 6S ) GlcNAc (6S ) 4GlcNAc( 6S ) B1- fucosylated . Heparin GICUA or GlcNAc or -4IdoUA ( 2S )al- Highest negative charge density of IdoUA (2S ) GICNS or 4GlcNS (6S )al any known biologicalmolecule GlcNAc (6S ) or GlcNS (6S ) Heparan GICUA or GlcNAc or -4GlcUAB1 Highly similar in structure to sulfate IdoUA or GICNS or 4GICNAcal heparin , however heparan sulfates IdoUA (2S ) GlcNAc (6S ) or disaccharide units are organised GlcNS (6 ) into distinct sulfated and non sulfated domains. US 10,722,444 B2 7 8 TABLE 1 - continued Examples of GAGS Glycosidic Hexuronic linkage Name acid /Hexose Hexosamine geometry Unique features Hyaluronan GlcUA GlcNAC -4G CUAB1 The only GAG that is exclusively 3GlcNAcB1 non - sulfated GlcUA = B - D - glucuronic acid GlcUA (2S ) = 2 - O - sulfo - B - D - glucuronic acid IdOUA = O - L - iduronic acid IdoUA (2S ) = 2-0 - sulfo -a -L - iduronic acid Gal = B - D - galactose Gal (68 ) = 6 - O - sulfo - B - D - galactose GalNAc = B - D -N -acetylgalactosamine GalNAc( 48 ) = B - D - N - acetylgalactosamine- 4-0 - sulfate GalNAc (6S ) = B -D - N -acetylgalactosamine - 6 -O - sulfate GalNAc( 48,6S ) = B - D - N -acetylgalactosamine -4-0 , 6 - O - sulfate GlcNAc = a - D - N - acetylglucosamine GlcNS = a - D - N - sulfoglucosamine GlcNS( 68 ) = a - D - N - sulfoglucosamine- 6 - O - sulfate Aspects of the present specification provide, in part, a ing B - 1,4 and B - 1,3 glycosidic bonds and pharmaceutically hydrogel composition comprising a chondroitin sulfate acceptable salts thereof. Hyaluronan polymers can be puri polymer . As used herein , the term “ chondroitin sulfate fied from animal and non -animal sources . Polymers of polymer ” refers to an unbranched , sulfated polymer of 25 hyaluronan can range in size from about 5,000 Da to about variable length comprising disaccharides of two alternating 20,000,000 Da. Any hyaluronan polymer is useful in the monosaccharides of D - glucuronic acid (GlcA ) and N - acetyl compositions disclosed herein with the proviso that the D -galactosamine (GalNAc ) and pharmaceutically accept hyaluronan improves a condition of the skin . Non - limiting able salts thereof. A chondroitin sulfate polymer may also examples of pharmaceutically acceptable salts of hyaluro include D - glucuronic acid residues that are epimerized into 30 nan include sodium hyaluronan , potassium hyaluronan , L -iduronic acid (IdoA ), in which case the resulting disac magnesium hyaluronan , calcium hyaluronan , and combina charide referred to as dermatan sulfate . A chondroitin tions thereof. sulfate polymer can have a chain of over 100 individual Aspects of the present specification provide , in part , a sugars, each of which can be sulfated in variable positions hydrogel composition comprising a crosslinked gly and quantities . Chondroitin sulfate polymers are an impor- 35 cosaminoglycan polymer . As used herein , the term “ cross tant structural component of cartilage and provide much of linked ” refers to the intermolecular bonds joining the indi its resistance to compression . Any chondroitin sulfate poly vidual polymer molecules , or monomer chains, into a more mer is useful in the compositions disclosed herein with the stable structure like a gel . As such , a crosslinked gly proviso that the chondroitin sulfate polymer improves a cosaminoglycan polymer has at least one intermolecular condition of the skin . Non - limiting examples of pharmaceu- 40 bond joining at least one individual polymer molecule to tically acceptable salts of chondroitin sulfate include sodium another one . The crosslinking of glycosaminoglycan poly chondroitin sulfate , potassium chondroitin sulfate , magne mers typically result in the formation of a hydrogel . Such sium chondroitin sulfate , calcium chondroitin sulfate , and hydrogels have high viscosity and require considerable force combinations thereof. to extrude through a fine needle . Aspects of the present specification provide , in part , a 45 Glycosaminoglycan polymers disclosed herein may be hydrogel composition comprising a keratan sulfate polymer . crosslinked using dialdehydes and disulfides crosslinking As used herein , the term “ keratan sulfate polymer ” refers to agents including , without limitation , multifunctional PEG a polymer of variable length comprising disaccharide units , based crosslinking agents , divinyl sulfones , diglycidyl which themselves include B - D -galactose and N - acetyl- D ethers, and bis - epoxides, biscarbodiimide. Non - limiting galactosamine (GalNAc ) and pharmaceutically acceptable 50 examples of hyaluronan crosslinking agents include multi salts thereof. Disaccharides within the repeating region of functional PEG -based crosslinking agents like pentaeryth keratan sulfate may be fucosylated and N - Acetylneuraminic ritol tetraglycidyl ether (PETGE ) , divinyl sulfone (DVS ) , acid caps the end of the chains. Any keratan sulfate polymer 1,4 -butanediol diglycidyl ether ( BDDE ) , 1,2 -bis ( 2,3 - epoxy is useful in the compositions disclosed herein with the propoxy ) ethylene (EGDGE ) , 1,2,7,8 - diepoxyoctane (DEO ) , proviso that the keratan sulfate polymer improves a condi- 55 (phenylenebis- ( ethyl) -carbodiimide and 1,6 hexamethylen tion of the skin . Non - limiting examples of pharmaceutically ebis ( ethylcarbodiimide) , adipic dihydrazide ( ADH ) , bis acceptable salts of keratan sulfate include sodium keratan ( sulfosuccinimidyl) suberate (BS ), hexamethylenediamine sulfate , potassium keratan sulfate , magnesium keratan sul (HMDA ), 1-( 2,3- epoxypropyl) -2,3 - epoxycyclohexane, or fate, calcium keratan sulfate, and combinations thereof. combinations thereof. Aspects of the present specification provide, in part , a 60 In accordance with the present specification , " % " in a hydrogel composition comprising a hyaluronan polymer. As formulation is defined as weight by weight (i.e. , w / w ) used herein , the term “ hyaluronic acid polymer ” is synony percentage . As an example : 1 % ( w / w ) means a concentration mous with “HA polymer” , “ hyaluronic acid polymer ” , and of 10 mg/ g. “ hyaluronate polymer ” refers to an anionic , non -sulfated In one aspect , a hydrogel composition is provided com glycosaminoglycan polymer comprising disaccharide units , 65 prising a crosslinked glycosaminoglycan polymer. For which themselves include D - glucuronic acid and D - N example , the composition may comprise a crosslinked chon acetylglucosamine monomers , linked together via alternat droitin sulfate polymer, a crosslinked dermatan sulfate poly US 10,722,444 B2 9 10 mer, a crosslinked keratan sulfate polymer , a crosslinked polymers include hyaluronan polymers of about 100,000 Da , heparan polymer , a crosslinked heparan sulfate polymer, or about 200,000 Da, about 300,000 Da, about 400,000 Da , a crosslinked hyaluronan polymer. The crosslinked gly about 500,000 Da , about 600,000 Da, about 700,000 Da , of cosaminoglycan may be present, e.g. , about 1 % by weight, about 800,000 Da, and about 900,000 Da. about 2 % by weight, about 3 % by weight, about 4 % by 5 In yet another embodiment , a composition comprises weight, about 5 % by weight, about 6 % by weight, about 7 % crosslinked hyaluronan polymers where the crosslinked by weight, about 8 % by weight, or about 9 % , or about 10 % hyaluronan polymers comprise a combination of both high by weight, of the total glycosaminoglycan present in the molecular weight hyaluronan polymers and low molecular composition . In yet other aspects of this embodiment , a weight hyaluronan polymers , in various ratios. In aspects of composition comprises a crosslinked glycosaminoglycan 10 this embodiment, a composition comprises crosslinked where the crosslinked glycosaminoglycan represents , e.g., at hyaluronan polymers where the crosslinked hyaluronan most 1 % by weight, atmost 2 % by weight, at most 3 % by polymers comprise a combination of both high molecular weight, at most 4 % by weight, at most 5 % by weight, atmost weight hyaluronan polymers and low molecular weight 6 % by weight, at most 7 % by weight, at most 8 % by weight, hyaluronan polymers in a ratio of about 20 : 1 , about 15 : 1 , at most 9 % by weight, or at most 10 % by weight, of the total 15 about 10 :1 , about 5 : 1, about 1: 1, about 1: 5 about 1:10 , about glycosaminoglycan present in the composition . In still other 1:15 , or about 1:20 . aspects of this embodiment, a composition comprises a Aspects of the present specification provide, in part , a crosslinked glycosaminoglycan where the crosslinked gly hydrogel composition comprising a crosslinked gly cosaminoglycan represents, e.g., about 0 % to about 20 % by cosaminoglycan polymer having a degree of crosslinking . weight, about 1 % to about 17 % by weight, about 3 % to 20 As used herein , the term “ degree of crosslinking ” refers to about 15 % by weight, or about 5 % to about 10 % by weight, the percentage of glycosaminoglycan polymer monomeric for example , about 11 % by weight, about 15 % by weight or units , such as, e.g. , the disaccharide monomer units of about 17 % by weight, of the total glycosaminoglycan pres hyaluronan that are bound to a cross- linking agent. The ent in the composition . degree of crosslinking is expressed as the percent weight The crosslinked glycosaminoglycan may be present in the 25 ratio of the crosslinking agent to glycosaminoglycan . composition at a concentration of, e.g. , about 2 mg/ g , about Aspects of the present specification provide , in part , a 3 mg/ g , about 4 mg/ g, about 5 mg/ g , about 6 mg/ g , about 7 hydrogel composition comprising an uncrosslinked gly mg/ g , about 8 mg/ g , about 9 mg/ g , about 10 mg/ g , about 11 cosaminoglycan polymer .As used herein , the term “ uncross mg/ g , about 12 mg/ g, about 13 mg/ g , about 13.5 mg/ g , about linked ” refers to a lack of intermolecular covalent bonds 14 mg/ g, about 15 mg/ g , about 16 mg/ g, about 17 mg/ g , 30 joining the individual glycosaminoglycan polymer mol about 18 mg/ g , about 19 mg/ g , or about 20 mg/ g , or greater, ecules, or monomer chains. As such , an uncrosslinked for example , up to about 40 mg/ g . In other aspects of this glycosaminoglycan polymer is not linked to any other embodiment, a composition comprises a crosslinked gly glycosaminoglycan polymer by an intermolecular bond . In cosaminoglycan where the crosslinked glycosaminoglycan aspects of this embodiment, a composition comprises an is present at a concentration of, e.g. , at least 1 mg/ g , at least 35 uncrosslinked chondroitin sulfate polymer, an uncrosslinked 2 mg/ g , at least 3 mg/ g , at least 4 mg/ g , at least 5 mg/ g , at dermatan sulfate polymer , an uncrosslinked keratan sulfate least 10 mg/ g , at least 15 mg/ g , at least 20 mg/ g , or at least polymer , an uncrosslinked heparan polymer , an uncross 25 mg/ g , or about 40 mg/ g . In yet other aspects of this linked heparan sulfate polymer , or an uncrosslinked hyaluro embodiment, a composition comprises a crosslinked gly nan polymer. Uncrosslinked glycosaminoglycan polymers cosaminoglycan where the crosslinked glycosaminoglycan 40 are water soluble and generally remain fluid in : nature . As is present at a concentration of, e.g., at most 1 mg/ g , at most such , uncross - linked glycosaminoglycan polymers are often 2 mg/ g , at most 3 mg/ g , at most 4 mg/ g , at . most 5 mg/ g , at mixed with a glycosaminoglycan polymer- based hydrogel most 10 mg/ g, at most 45 mg/ g , atmost 20 mg/ g, at most 25 composition as a lubricant to facilitate the extrusion process mg/ g , or at most 40 mg/ g . In still other aspects of this of the composition through a fine needle . embodiment, a composition comprises a crosslinked gly- 45 In an embodiment, a composition comprises an uncross cosaminoglycan where the crosslinked glycosaminoglycan linked glycosaminoglycan polymer where the uncrosslinked is present at a concentration of , e.g., about 7.5 mg/ g to about glycosaminoglycan polymer is present in an amount suffi 19.5 mg/ g , about 8.5 mg/ g to about 18.5 mg/ g , about 9.5 cient to improve a skin condition as disclosed herein . In mg/ g to about 17.5 mg/ g , about 10.5 mg/ g to about 16.5 aspects of this embodiment, a composition comprises an mg/ g , about 11.5 mg/ g to about 15.5 mg/ g , or about 12.5 50 uncrosslinked glycosaminoglycan where the uncrosslinked mg/ g to about 14.5 mg/ g , up to about 40 mg/ g . glycosaminoglycan is present at a concentration of, e.g. , Aspects of the present specification provide , in part , a about 2 mg/ g , about 3 mg/ g, about 4 mg/ g , about 5 mg/ g , hydrogel composition comprising hyaluronan polymers of about 6 mg/ g , about 7 mg/ g , about 8 mg/ g , about 9 mg/ g , low molecular weight, hyaluronan polymers of high molecu about 10 mg/ g , about 11 mg/ g , about 12 mg/ g , about 13 lar weight, or hyaluronan polymers of both low and high 55 mg/ g, about 13.5 mg/ g , about 14 mg/ g , about 15 mg/ g , about molecular weight. As used herein , the term “ high molecular 16 mg/ g , about 17 mg/ g , about 18 mg/ g , about 19 mg/ g , weight" when referring to " hyaluronan ” refers to hyaluronan about 20 mg/ g , about 40 mg/ g , or about 60 mg/ g . In other polymers having a mean molecular weight of 1,000,000 Da aspects of this embodiment, a composition comprises an or greater. Non - limiting examples of a high molecular uncrosslinked glycosaminoglycan where the uncrosslinked weight hyaluronan polymers include hyaluronan polymers 60 glycosaminoglycan is present at a concentration of, e.g., at about 1,500,000 Da, about 2,000,000 Da , about 2,500,000 least 1 mg/ g, at least 2 mg/ g , at least 3 mg/ g , at least 4 mg/ g , Da, about 3,000,000 Da , about 3,500,000 Da, about 4,000 , at least 5 mg/ g , at least 10 mg/ g , at least 15 mg/ g , at least 20 000 Da, about 4,500,000 Da, and about 5,000,000 Da. As mg/ g, at least 25 mg/ g at least 35 mg/ g , or at least 40 mg/ g . used herein , the term “ low molecular weight ” when referring In yet other aspects of this embodiment, a composition to “ hyaluronan ” refers to hyaluronan polymers having a 65 comprises an uncrosslinked glycosaminoglycan where the mean molecular weight of less than 1,000,000 Da. Non uncrosslinked glycosaminoglycan is present at a concentra limiting examples of a low molecular weight hyaluronan tion of, e.g. , at most 1 mg / g, at most 2 mg/ g, at most 3 mg/ g , US 10,722,444 B2 11 12 at most 4 mg/ g , at most 5 mg/ g , at most 10 mg/ g , at most 15 hyaluronan polymers , in various ratios . In aspects of this mg/ g , at most 20 mg/ g , or at most 25 mg/ g . In still other embodiment, a composition comprises an uncrosslinked aspects of this embodiment, a composition comprises an hyaluronan polymers where the uncrosslinked hyaluronan uncrosslinked glycosaminoglycan where the uncrosslinked polymers comprises a combination of both high molecular glycosaminoglycan is present at a concentration of, e.g., 5 weight hyaluronan polymers and low molecular weight about 1 mg / g to about 60 mg/ g , about 10 mg/ g to about 40 hyaluronan polymers in a ratio of about 20 : 1, about 15 : 1, mg/ g , about 7.5 mg/ g to about 19.5 mg/ g , about 8.5 mg/ g to about 10 : 1 , about 5 : 1 , about 1: 1 , about 1 :5 about 1:10 , about about 18.5 mg/ g , about 9.5 mg/ g to about 17.5 mg/ g , about 1:15 , or about 1:20 . 10.5 mg/ g to about 16.5 mg/ g , about 11.5 mg/ g to about 15.5 Aspects of the present specification provide , in part, a mg/ g , or about 12.5 mg/ g to about 14.5 mg/ g . 10 hydrogel composition comprising a substantially uncross In an embodiment, a composition comprises uncross linked glycosaminoglycan polymer . As sued herein , the term linked hyaluronan polymers of low molecular weight. In “ substantially uncrosslinked ” refers to the presence of aspects of this embodiment, a composition comprises a uncrosslinked glycosaminoglycan polymers in a composi uncrosslinked hyaluronan having a mean molecular weight tion disclosed herein at a level of at least 90 % by weight of of, e.g., about 100,000 Da, about 200,000 Da, about 300,000 15 the composition , with the remaining at most 10 % by weight Da, about 400,000 Da, about 500,000 Da, about 600,000 Da, of the composition being comprised of other components about 700,000 Da, about 800,000 Da, or about 900,000 Da . including crosslinked glycosaminoglycan polymers . In In yet other aspects of this embodiment, a composition aspects of this embodiment, a composition comprises a comprises uncrosslinked hyaluronan polymers having a substantially uncrosslinked chondroitin sulfate polymer , a mean molecular weightof , e.g., at most 100,000 Da , at most 20 substantially uncrosslinked dermatan sulfate polymer , a sub 200,000 Da , at most 300,000 Da, at most 400,000 Da , at stantially uncrosslinked keratan sulfate polymer , a substan most 500,000 Da , at most 600,000 Da , at most 700,000 Da, tially uncrosslinked heparan polymer, a substantially atmost 800,000 Da, at most 900,000 Da, or at most 950,000 . uncrosslinked heparan sulfate polymer , or a substantially In still other aspects of this embodiment, a composition uncrosslinked hyaluronan polymer. In other aspects of this comprises uncrosslinked hyaluronan polymers having a 25 embodiment, a composition comprises an uncrosslinked mean molecular weight of, e.g. , about 100,000 Da to about glycosaminoglycan where the uncrosslinked glycosamino 500,000 Da , about 200,000 Da to about 500,000 Da, about glycan represents , e.g., about 90 % or more by weight, about 300,000 Da to about 500,000 Da , about 400,000 Da to about 91 % or more by weight, about 92 % or more by weight, 500,000 Da , about 500,000 Da to about 950,000 Da, about about 93 % or more by weight, about 94 % or more by 600,000 Da to about 950,000 Da, about 700,000 Da to about 30 weight, about 95 % or more by weight, about 96 % or more 950,000 Da , about 800,000 Da to about 950,000 Da, about by weight, about 97 % or more by weight, about 98 % or 300,000 Da to about 600,000 Da, about 300,000 Da to about more by weight, or about 99 % or more, or about 100 % by 000 Da, about 300,000 Da to about 800,000 Da , or weight, of the total glycosaminoglycan present in the com about 400,000 Da to about 700,000 Da. position . In yet other aspects of this embodiment, a com In another embodiment, a composition comprises 35 position comprises an uncrosslinked glycosaminoglycan uncrosslinked hyaluronan polymers of high molecular where the uncrosslinked glycosaminoglycan represents , e.g. , weight. In aspects of this embodiment, a composition com about 90 % to about 100 % by weight, about 93 % to about prises an uncrosslinked hyaluronan having a mean molecu 100 % by weight, about 95 % to about 100 % by weight, or lar weight of, e.g. , about 1,000,000 Da , about 1,500,000 Da , about 97 % to about 100 % by weight, of the total gly about 2,000,000 Da, about 2,500,000 Da, about 3,000,000 40 cosaminoglycan present in the composition . Da, about 3,500,000 Da, about 4,000,000 Da, about 4,500 , Aspects of the present specification provide, in part , a 000 Da , or about 5,000,000 Da. In other aspects of this hydrogel composition comprising a ratio of crosslinked embodiment, a composition comprises an uncrosslinked glycosaminoglycan polymer and uncrosslinked gly hyaluronan polymers having a mean molecular weight of, cosaminoglycan polymer. This ratio of crosslinked and e.g., at least 1,000,000 Da , at least 1,500,000 Da , at least 45 uncrosslinked glycosaminoglycan polymer is also known as 2,000,000 Da, at least 2,500,000 Da, at least 3,000,000 Da , the gel: fluid ratio . Any gel : fluid ratio is useful in making the at least 3,500,000 Da, at least 4,000,000 Da, at least 4,500 , compositions disclosed herein with the proviso that such 000 Da , or at least 5,000,000 Da . In yet other aspects of this ratio produces a composition disclosed herein that improves embodiment, a composition comprises an uncrosslinked a skin condition as disclosed herein . Non - limiting examples hyaluronan polymers having a mean molecular weight of, 50 of gel : fluid ratios include 100 :0 , 98 : 2 , 90:10 , 75:25 , 70:30 , e.g., about 1,000,000 Da to about 5,000,000 Da, about 60:40 , 50:50 , 40:60 , 30:70 , 25:75 , 10:90 ; 2:98 , and 0 : 100 . 1,500,000 Da to about 5,000,000 Da, about 2,000.000 Da to In aspects of this embodiment, a composition comprises about 5,000,000 Da, about 2,500,000 Da to about 5,000,000 a crosslinked glycosaminoglycan polymer and an uncross Da, about 2,000,000 Da to about 3,000,000 Da , about linked glycosaminoglycan polymer where the gel: fluid ratio 2,500,000 Da to about 3,500,000 Da, or about 2,000,000 Da 55 is , e.g., about 0 : 100 , about 1:99 , about 2:98 , about 3:97 , to about 4,000,000 Da . In still other aspects , a composition about 4:96 , about 5:95 , about 6:94 , about 7:93 , about 8:92 , comprises an uncrosslinked hyaluronan polymers having a about 9:91, or about 10:90 . In other aspects of this embodi mean molecular weight of, e.g., greater than 2,000,000 Da ment, a composition comprises a crosslinked glycosamino and less than about 3,000,000 Da, greater than 2,000,000 Da glycan polymer and an uncrosslinked glycosaminoglycan and less than about 3,500,000 Da, greater than 2,000,000 Da 60 polymer where the gel : fluid ratio is , e.g., at most 1:99, at and less than about 4,000,000 Da, greater than 2,000,000 Da most 2:98 , at most 3:97 , at most 4:96 , at most 5:95 , at most and less than about 4,500,000 Da , greater than 2,000,000 Da 6:94 , at most 7:93 , at most 8:92 , at most 9:91 , or at most and less than about 5,000,000 Da . 10:90 . In yet other aspects of this embodiment, a composi In another embodiment, a composition comprises tion comprises a crosslinked glycosaminoglycan polymer uncrosslinked hyaluronan polymers where the uncrosslinked 65 and an uncrosslinked glycosaminoglycan polymer where the hyaluronan comprises a combination ofboth high molecular gel : fluid ratio is , e.g. , about 0 : 100 to about 3:97 , about 0 : 100 weight hyaluronan polymers and low molecular weight to about 5:95 , or about 0 : 100 to about 10:90 . US 10,722,444 B2 13 In other aspects of this embodiment, a composition com well known to have diverse beneficial biochemical and prises a crosslinked glycosaminoglycan polymer and an antioxidant effects . Non - limiting examples of flavonoids uncrosslinked glycosaminoglycan polymer where the gel : include C - methylated flavonoids, O -methylated flavonoids, fluid ratio is , e.g., about 15:85 , about 20:80 , about 25:75 , isoflavonoids, neoflavonoids, flavonolignans, furanofla about 30:70 , about 35:65 , about 40:60 , about 45:55, about 5 vonoids, pyranoflavonoids ,methylenedioxyflavonoids , pre 50:50 , about 55:45 , about 60:40 , about 65:35 , about 70:30 , nylated flavonoids, aurones, flavones, flavonols , flavanones, about 75:25 , about 80:20 , about 85:15 , about 90:10 , about 95 : 5 , about 98 : 2 , or about 100 : 0 . In yet other aspects of this flavanonols , flavan - 3 - ols , flavan - 4 - ols , leucoanthocyanidin embodiment, a composition comprises a crosslinked gly ( flavan - 3,4 - diols ), anthocyanidins , and tannins. cosaminoglycan polymer and an uncrosslinked gly- 10 In some preferred embodiments , the antioxidant agent is cosaminoglycan polymer where the gel: fluid ratio is , e.g., at a water soluble flavonoid , for example , a glucosylated most 15:85 , at most 20:80 , at most 25:75 , at most 30:70 , at flavonoid , for example, Epigallocatechin Gallatyl Glucoside most 35:65 , at most 40:60 , at most 45:55 , at most 50:50 , at (e.g. Inoveol® EGCG ) . most 55:45 , at most 60:40 , at most 65:35 , at most 70:30 , at Aurones are flavonoid compounds derived from 2 -ben most 75:25 , at most 80:20 , at most 85:15 , at most 90:10 , at 15 zylidene- 1 -benzofuran - 3 - one. Non -limiting examples of most 95 : 5 , at most 98 : 2 , or at most 100 : 0 . In still other aurones include 4,5,6 - trihydroxy -aurone , aureusidin , hispi aspects of this embodiment, a composition comprises a dol, leptosidin , maritimetin , and sulfuretin . crosslinked glycosaminoglycan polymer and an uncross Three major classes of ketone -containing flavonoids are linked glycosaminoglycan polymer where the gel: fluid ratio flavones, compounds derived from 2 -phenylchromen - 4 -one is , e.g., about 10:90 to about 70:30 , about 15:85 to about 20 ( 2 - phenyl- 1,4 -benzopyrone ) ; isoflavones, compounds 70:30 , about 10:90 to about 55:45 , about 80:20 to about derived from 3 - phenylchromen - 4 -one ( 3 - phenyl- 1,4 -benzo 95 : 5 , about 90:10 to about 100 : 0 , about 75:25 to about pyrone ); and neoflavones , compounds derived from 4 -phe 100 : 0 , or about 60:40 to about 100 : 0 . nylcoumarine (4 -phenyl - 1,2 -benzopyrone ) ( Table 2 ). Fla Aspects of the present specification provide, in part, a vones are themselves divided into four groups based on the hydrogel composition disclosed herein that may optionally 25 presence or absence of 3- hydroxyl 2,3 -dihydro functional comprise an anesthetic agent. An anesthetic agent is pref groups : flavones, compounds derived from 2- phenyl erably a local anesthetic agent, i.e. , an anesthetic agent that chromen - 4 -one lack both functional groups; flavonols ( 3 -hy causes a reversible local anesthesia and a loss of nocicep droxyflavone ), compounds derived from 3 -hydroxy - 2 -phe tion , such as, e.g., aminoamide local anesthetics and amin nylchromen - 4 -one have the 3 -hydroxyl group , but lack the oester local anesthetics . The amount of an anesthetic agent 30 2,3 -dihydro group ; flavanones , compounds derived from included in a composition disclosed herein is an amount 2,3 - dihydro - 2 - phenylchromen - 4 -one have the 2,3 -dihydro effective to mitigate pain experienced by an individual upon group , but lack the 3 -hydroxyl group ; and flavanonols administration of the composition . As such , the amountof an ( 3 -hydroxyflavanone or 2,3 -dihydroflavonol ) , compounds anesthetic agent included in a composition disclosed in the derived from 3 -hydroxy - 2,3 - dihydro - 2 - phenylchromen - 4 present specification is between about 0.1 % to about 5 % by 35 one have both functional groups. weight of the total composition The anesthetic agent may be Non -limiting examples of flavones include acacetin , any suitable anesthetic agent, for example , but not limited to apiin , apigenin , apigetrin , artoindonesianin P, baicalein , lidocaine. A composition disclosed herein may comprise a baicalin , chrysin , cynaroside, diosmetin , diosmin , eupatilin , single anesthetic agent or a plurality of anesthetic agents . A flavoxate , 6 -hydroxyflavone , genkwanin , hidrosmin , luteo non - limiting example of a combination local anesthetic is 40 lin , nepetin , nepitrin ( nepetin 7 - glucoside ), nobiletin , orien lidocaine/ prilocaine (EMLA ) . tin ( isoorientin ), oroxindin , oroxylin A , rhoifolin , scutella The anesthetic agentmay be present in the composition in rein , scutellarin , tangeritin , techtochrysin , tetuin , tricin , an amount of, e.g., about 0.1 % , about 0.2 % , about 0.3 % , veronicastroside, vitexin ( isovitexin ), and wogonin . Non about 0.4 % , about 0.5 % , about 0.6 % , about 0.7 % , about limiting examples of flavonols include 3 - hydroxyflavone , 0.8 % about 0.9 % , about 1.0 % , about 2.0 % , about 3.0 % , 45 azaleatin , fisetin , galangin , gossypetin , kaempferide , kaemp about 4.0 % , about 5.0 % . , or more , by weight of the total ferol, isorhamnetin , morin , myricetin , natsudaidain , pachy composition . In further aspects , a composition disclosed podol, quercetin , rhamnazin , rhamnetin , and sophorin . Non herein comprises an anesthetic agent in an amount of , e.g. , limiting examples of flavanones include butin , eriodictyol, about 0.1 % to about 0.5 % , about 0.1 % to about 1.0 % , about hesperetin , hesperidin , homoeriodictyol, isosakuranetin , 0.1 % to about 2.0 % , about 0.1 % to about 3.0 % , about 0.1 % 50 naringenin , naringin , pinocembrin , poncirin , sakuranetin , to about 4.0 % , about 0.1 % to about 5.0 % , about 0.2 % to sakuranin , and sterubin . Non - limiting examples of flavanon about 0.9 % , about 0.2 % to about 1.0 % , about 0.2 % to about ols include taxifolin ( dihydroquercetin ), and aromadedrin 2.0 % , about 0.5 % to about 1.0 % , or about 0.5 % to about (dihydrokaempferol ). 2.0 % by weight of the total composition . Isoflavonoids include isoflavones and isoflavanes ( Table The amount of an anti -oxidant agent included in a com- 55 2 ). Non -limiting examples of isoflavonoids include alpinu position disclosed herein is an amount effective to reduce or misoflavone , anagyroidisoflavone A and B , calycosin , daid prevent degradation of the composition , such as , e.g., oxi zein , daidzin , derrubone , di- O -methylalpinumisoflavone , dative stresses , enzymatic degradation and /or chemical deg formononetin , genistein , genistin , glycitein , ipriflavone, iri radation of the composition . The amount of an anti -oxidant genin , iridin , irilone, 4 '- methyl - alpinumisoflavone , 5-0 agent included in a composition disclosed herein may be 60 methylgenistein , luteone , ononin , orobol , pratensein , prune between about 0.1 % to about 10 % by weight of the total tin , pseudobaptigenin , psi- tectorigenin , puerarin , retusin , composition . tectoridin , tectorigenin , and wighteone . Aspects of the present specification provide , in part , a Neoflavonoids include 4 -arylcoumarins (neoflavones ) , hydrogel composition disclosed herein that may comprise a 4 -arylchromanes , dalbergiones and dalbergiquinols ( Table flavonoid ( Table 2) . A flavonoid (or bioflavonoid ) refers to 65 2) . Neoflavones are compounds derived from 4 -phenylcou the class of polyphenolic ketone -containing and non -ketone marin (or 4 - Aryl- coumarin ); neoflavenes compounds containing secondary metabolites found in plants that are derived from 4 -phenylchromen . Non - limiting examples of US 10,722,444 B2 15 16 neoflavonoids include calophyllolide , coutareagenin , dalber terflavins B , tergallagin , vescalin , 1,3,4 - tri- O -galloylquinic gichromene , dalbergin , and nivetin . acid , 3,5 -di - O -galloyl - shikimic acid , and 3,4,5 - tri - O - gal Non -ketone - containing flavonoids include flavan -3 -ols loylshikimic acid . and catechins. Flavan - 3 -ols ( flavanols ) are a class of fla Condensed tannins (proanthocyanidins ) are essentially vonoids derived from 2 - phenyl- 3,4 -dihydro -2H - chromen -3- 5 polymer chains of flavonoids such as catechins. Non - limit ol skeleton . Catechin possesses two benzene rings (called ing examples of condensed tannins include proanthocyani the A- and B - rings ) and a dihydropyran heterocycle ( the din , prodelphinidin , profisetinidin , proguibourtinidin , and C - ring ) with an hydroxyl group on carbon 3. The A ring is prorobinetidin . similar to a resorcinol moiety while the B ring is similar to a catechol moiety . There are two chiral centers on the 10 TABLE 2 molecule on carbons 2 and 3. It has therefore four diaste Flavonoids reoisomers . Two of the isomers are in trans configuration and are called catechin and the other two are in cis configu Flavonoids Base compound Examples ration and are called epicatechin . Non -limiting examples of 15 Aurones 2 -benzylidene - 1 4,5,6 -trihydroxy -aurone , non -ketone -containing flavonoids include afzelechin , benzofuran - 3 -one aureusidin , hispidol, leptosidin , maritimetin , and sulfuretin arthromerin A , arthromerin B , catechin , epicatechin , epigal Flavones 2- phenylchromen acacetin , apiin , apigenin , apigetrin , locatechin , epicatechin gallate , epigallocatechin gallate , epi 4 - one artoindonesianin P , baicalein , gallocatechin gallate, epiafzelechin , fisetinidol, gallocat baicalin , chrysin , cynaroside , diosmetin , diosmin , eupatilin , echin , gallocatechin gallate , guibourtinidol , meciadanol 20 flavoxate , 6 - hydroxyflavone , ( 3 - O -methylcatechin ), mesquitol, propyl gallate , robinetini genkwanin , hidrosmin , luteolin , dol, and thearubigin . nepetin , nepitrin , nobiletin , orientin , oroxindin , oroxylin A , rhoifolin , Flavan - 4 -ols ( 3 -deoxyflavonoids ) are flavone -derived scutellarein , scutellarin , tangeritin , alcohols derived from 2 - phenylchroman - 4 - ol. Non - limiting techtochrysin , tetuin , tricin , examples of flavan - 4 -ols include apiforol and luteoforol. 25 veronicastroside, vitexin , wogonin Flavonols 3 -hydroxy - 2 -phenyl 3 -hydroxyflavone , azaleatin , fisetin , Leucoanthocyanidin ( flavan - 3,4 -diols ) are compounds chromen - 4 - one galangin , gossypetin , kaempferide , derived from 2 -phenyl - 3,4 - dihydro - 2H -chromene - 3,4 - diol. kaempferol, isorhamnetin , morin , Non -limiting examples of flavan -3,4 -diols include leucocya myricetin , natsudaidain , nidin , leucodelphinidin , leucomalvidin , leucopelargonidin , pachypodol, quercetin , rhamnazin , 30 rhamnetin , sophorin leucopeonidin , leucorobinetinidin , and melacacidin . Flavanones 2,3 - dihydro - 2 -phenyl- butin , eriodictyol , hesperetin , Anthocyanidins are compounds derived from 2 -phenyl chromen - 4 -one hesperidin , homoeriodictyol , chromenylium . Non - limiting examples of anthocyanidins isosakuranetin , naringenin , include antirrhinin , apigeninidin , aurantinidin , capensinidin , naringin , pinocembrin , poncirin , chrysanthenin , columnidin , commelinin , cyanidin , 6 -hy- 35 Flavanonols 3- hydroxy -2,3 -dihydro- sakuranetinaromadedrin , ,sakuranin taxifolin , sterubin droxycyanidin , cyanidin - 3 - di -p -coumarylglucoside ) -5 - glu 2 -phenylchromen 4 - one coside, cyanosalvianin , delphinidin , diosmetinidin , euro Isoflavones 3 - phenylchromen alpinumisoflavone , pinidin , fisetinidin , gesneridin , guibourtinidin , hirsutidin , 4 - one anagyroidisoflavone A and B , luteolinidin , malvidin , 5 -desoxy -malvidin , malvin , myrtil calycosin , daidzein , daidzin , derrubone, di- O lin , oenin , peonidin , 5- desoxy -peonidin , pelargonidin , petu- 40 methylalpinumisoflavone, nidin , primulin , protocyanin , protodelphin , pulchellidin , formononetin , genistein , genistin , pulchellidin 3 - glucoside, pulchellidin 3- rhamnoside , robin glycitein , ipriflavone , irigenin , iridin , irilone, 4 '- methyl etinidin , rosinidin , tricetinidin , tulipanin , and violdelphin . alpinumisoflavone , 5-0 Tannins are compounds derived from 2 -phenylchromeny methylgenistein , luteone, ononin , lium . There are three major classes of tannins: hydrolyzable 45 orobol, pratensein , prunetin , tannins ; non -hydrolyzable tannins (condensed tannins ; pseudobaptigenin , psi- tectorigenin , puerarin , retusin , tectoridin , proanthocyanidins ); and pseudotannins. tectorigenin , wighteone Hydrolyzable tannins are themselves divided into four Isoflavenes 3 -phenylchroman lonchocarpane , laxiflorane groups: oligomer tannins including aglycone tannins and Neoflavones 4 - phenylcoumarine calophyllolide glycoside tannins; ellagitannins ; gallotannins , and unclassi- 50 Neoflavenes 4 - phenylchromen dalbergichromene Flavan - 3 - ols 2 -phenyl - 3,4 - dihydro- arthromerin A , arthromerin B , fied tannins. Non -limiting examples of aglycone tannins 2H - chromen - 3 - ol fisetinidol, guibourtinidol , include ellagic acid , gallagic acid , and gallic acid . Non meciadanol ( 3 -O -methylcatechin ), limiting examples of glycoside tannins include glucose , mesquitol, robinetinidol, quinic acid , and shikimic acid . Non - limiting examples of thearubigin . Catechins (2R ,3S )-2- ( 3,4 - dihydro- ( + )- catechin ( 2R - 3S ), (- )- catechin ellagitannins include castalagin (vescalagin ), castalin , casu- 55 xyphenyl) -3,4 -dihydro- (2S - 3R ), ( - ) - Epicatechin (2R - 3R ), arictin , casuariin , casuarinin , cornusiin E , grandinin , pedun 2H -chromene - 3,5,7 ( + ) -epicatechin (2S - 3S ) culagin , punicacortein C , punigluconin , punicalagin , puni triol calagin alpha , punicalin , 2-0 - galloyl- punicalin , stachyurin , Flavan - 4 2 -phenylchroman - 4 - ol apiforol, luteoforol ols strictinin , and tellimagrandin II. Non - limiting examples of Flavan - 3,4 2 -phenyl - 3,4 - dihydro- leucocyanidin , leucodelphinidin , gallotannins include corilagin , galloyl glucose, digalloyl 60 diols 2H - chromene- 3,4 -diol leucomalvidin , leucopelargonidin , glucose, trigalloyl glucose , tetragalloyl glucose , pentagal leucopeonidin, leucorobinetinidin , loyl glucose , hexagalloyl glucose , heptagalloyl glucose , melacacidin Antho 2 -phenylchromenylium antirrhinin , apigeninidin , octagalloyl glucose , and tannic acid . Non - limiting examples cyanidins aurantinidin , capensinidin , of unclassified tannins include acutissimin A , acutissimin B , chrysanthenin , columnidin , chebulagic acid , chebulinic acid , cinnamtannin B1, combre- 65 commelinin , cyanidin , 6 -hydro glutinin , geraniin , granatin B , roburin A , roburin B , roburin xycyanidin , cyanidin - 3- (di - p C , roburin D , roburin E , stachyurin , tercatin , terflavins A , US 10,722,444 B2 17 18 TABLE 2 - continued total composition . In further aspects , a composition dis closed herein comprises an antioxidant agent in an amount Flavonoids of, e.g. , about 0.1 % to about 0.5 % , about 0.1 % to about Examples 1.0 % , about 0.1 % to about 2.0 % , about 0.1 % to about 3.0 % , Flavonoids Base compound 5 about 0.1 % to about 4.0 % , about 0.1 % to about 5.0 % , about coumarylglucoside ) -5 - glucoside , 0.2 % to about 0.9 % , about 0.2 % to about 1.0 % , about 0.2 % cyanosalvianin , delphinidin , diosmetinidin , europinidin , to about 2.0 % , about 0.5 % to about 1.0 % , or about 0.5 % to fisetinidin , gesneridin , about 2.0 % by weight of the total composition . guibourtinidin , hirsutidin , Aspects of the present specification provide, in part , a luteolinidin , malvidin , 5 -desoxy 10 hydrogel composition disclosed herein that exhibits a com malvidin , malvin , myrtillin , oenin , peonidin , 5 -desoxy -peonidin , plex modulus, an elastic modulus, a viscous modulus and /or pelargonidin , petunidin , primulin , a tan d . The compositions as disclosed herein are viscoelas protocyanin , protodelphin , tic in that the composition has an elastic component ( solid pulchellidin , pulchellidin 3 glucoside , pulchellidin 3 like such as , e.g., crosslinked glycosaminoglycan polymers) rhamnoside , robinetinidin , 15 and a viscous component (liquid - like such as , e.g., uncross rosinidin , tricetinidin , linked glycosaminoglycan polymers or a carrier phase ) tulipanin , violdelphin when a force is applied ( stress , deformation ). The rheologi Hydro gallic acid or ellagic castalagin , castalin , casuari?tin , cal attribute that described this property is the complex lyzable acid casuariin , casuarinin , corilagin , tannins cornusiin E , grandinin , galloyl modulus (G * ), which defines a composition's total resis glucose , digalloyl glucose , 20 tance to deformation . The complex modulus is a complex trigalloyl glucose , tetra number with a real and imaginary part: G * = G ' + iG " . The galloyl glucose , pentagalloyl glucose, hexagalloyl absolute value of G * is Abs (G * ) = Sqrt ( G 2 + G " 2 ). The com glucose, heptagalloyl glucose , plex modulus can be defined as the sum of the elastic octagalloyl glucose , pedunculagin , modulus ( G ') and the viscous modulus ( G " ) . punicacortein C , punigluconin , 25 The complex modulus describes the interaction between punicalagin , punicalagin alpha , elasticity and strength ( G * = stress/ strain ) and , as such , pro punicalin, 2-0 - galloyl- punicalin , stachyurin , strictinin , tannic acid , vides a quantitative measurement of a composition's hard tellimagrandin II ness or softness ; stress is the force causing the deformation Condensed polymer chains of proanthocyanidin , prodelphinidin , divided by the area to which the force is applied ; and strain tannins flavonoid units profisetinidin , proguibourtinidin , 30 is the ratio of the change caused by the stress to the original prorobinetidin state of the object. Although depending on the speed at which the force is applied , a stiffer composition will have a Aspects of the present specification provide , in part , a higher complex modulus ( and usually a higher elastic modu hydrogel composition disclosed herein that may optionally lus in the case of crosslinked fillers ) and it will take a greater comprise a flavonoid - type phytoalexin . A phytoalexin refers 35 force to deform the material a given distance , such as , e.g. , an injection . Complex modulus can be decomposed into an to the class of antimicrobial molecules with antioxidant elastic modulus and a viscous modulus , reflecting the fact effects synthesized de novo by plants in response to an that the compositions of the invention have viscoelasric incompatible pathogen infection . Non - limiting examples of properties . Elastic modulus, or modulus of elasticity , refers phytoalexins include resveratrol ( 3,5,4 '- trihydroxy -trans 40 to the ability of a hydrogel material to recover from shear stilbene) , allixin (3 -hydroxy - 5 -methoxy - 6 -methyl - 2 -pentyl deformation , or , conversely , an object's tendency to be 4H -pyran - 4 -one ) , glyceollin , phaseolin , and medicarpin . non -permanently deformed when a force is applied to it. In an embodiment, a composition disclosed herein com Elastic modulus characterizes the elastic recovery of a prises at least one antioxidant agent in an amount sufficient composition and is also known as the storage modulus to reduce or prevent degradation of a glycosaminoglycan 45 because it describes how much energy is stored upon shear polymer. deformation of the composition . In other aspects of this embodiment, a composition dis Viscous modulus is also known as the loss modulus closed herein comprises an antioxidant agent in an amount because it describes the energy that is lost as heat dissipation of, e.g., about 0.01 % , about 0.1 % , about 0.2 % , about 0.3 % , during shear deformation . Tan d is the ratio of the viscous about 0.4 % , about 0.5 % , about 0.6 % , about 0.7 % , about 50 modulus and the elastic modulus, tan d = G " /G '. For tan d 0.8 % about 0.9 % , about 1.0 % , about 2.0 % , about 3.0 % , values disclosed in the present specification , a tan d is about 4.0 % , about 5.0 % , about 6.0 % , about 7.0 % , about obtained from the elastic / viscous modulus at a frequency of 8.0 % , about 9.0 % , or about 10 % by weight of the total 1 Hz. A lower tan d corresponds to a more elastic compo composition . In yet other aspects , a composition disclosed sition . For fillers described in this invention , complex , herein comprises an antioxidant agent in an amount of, e.g. , 55 elastic and viscous modulus are usually measured through at least 0.1 % , at least 0.2 % , at least 0.3 % , at least 0.4 % , at shear deformation in oscillation mode (with a sinusoidal least 0.5 % , at least 0.6 % , at least 0.7 % , at least 0.8 % at least input and response ). 0.9 % , at least 1.0 % , at least 2.0 % , at least 3.0 % , at least In another embodiment, a hydrogel composition disclosed 4.0 % , at least 5.0 % , at least 6.0 % , at least 7.0 % , at least herein exhibits an elastic modulus. In aspects of this embodi 8.0 % , at least 9.0 % , or at least 10 % by weight of the total 60 ment, a hydrogel composition exhibits an elastic modulus of , composition . In still other aspects , a composition disclosed e.g., about 25 Pa , about 50 Pa , about 75 Pa , about 100 Pa , herein comprises an antioxidant agent in an amount of, e.g., about 125 Pa , about 150 Pa , about 175 Pa , about 200 Pa , atmost 0.1 % , at most 0.2 % , at most 0.3 % , at most 0.4 % , at about 250 Pa , about 300 Pa , about 350 Pa , about 400 Pa , most 0.5 % , at most 0.6 % , at most 0.7 % , at most 0.8 % at about 450 Pa , about 500 Pa, about 550 Pa , about 600 Pa , most 0.9 % , at most 1.0 % , at most 2.0 % , at most 3.0 % , at 65 about 650 Pa , about 700 Pa , about 750 Pa, about 800 Pa , most 4.0 % , at most 5.0 % , at most 6.0 % , at most 7.0 % , at about 850 Pa, about 900 Pa , about 950 Pa, about 1,000 Pa , most 8.0 % , at most 9.0 % , or atmost 10 % by weight of the about 1,200 Pa, about 1,300 Pa , about 1,400 Pa , about 1,500 US 10,722,444 B2 19 20 Pa, about 1,600 Pa , about 1700 Pa , about 1800 Pa , about Pa to about 350 Pa, about 300 Pa to about 400 Pa , about 350 1900 Pa, about 2,000 Pa , about 2,100 Pa , about 2,200 Pa , Pa to about 450 Pa, about 400 Pa to about 500 Pa , about 450 about 2,300 Pa , about 2,400 Pa , or about 2,500 Pa . In other Pa to about 550 Pa , about 500 Pa to about 600 Pa , about 550 aspects of this embodiment, a hydrogel composition exhibits Pa to about 650 Pa , or about 600 Pa to about 700 Pa . an elastic modulus of, e.g. , at least 25 Pa , at least 50 Pa , at ui In another embodiment, a hydrogel composition disclosed least 75 Pa , at least 100 Pa , at least 125 Pa , at least 150 Pa , herein exhibits a tan 8. In aspects of this embodiment, a at least 175 Pa , at least 200 Pa , at least 250 Pa , at least 300 hydrogel composition exhibits a tan d of, e.g. , about 0.1, Pa , at least 350 Pa , at least 400 Pa, at least 450 Pa, at least about 0.2 , about 0.3 , about 0.4 , about 0.5 , about 0.6 , about 500 Pa , at least 550 Pa , at least 600 Pa , at least 650 Pa , at 0.7 , about 0.8 , about 0.9 , about 1.0 , about 1.1, about 1.2 , least 700 Pa , at least 750 Pa , at least 800 Pa , at least 850 Pa , 10 about 1.3 , about 1.4 , about 1.5 , about 1.6 , about 1.7 , about at least 900 Pa , at least 950 Pa , at least 1,000 Pa , at least 1.8 , about 1.9, about 2.0 , about 2.1 , about 2.2 , about 2.3 , 1,200 Pa , at least 1,300 Pa , at least 1,400 Pa , at least 1,500 about 2.4 , or about 2.5 . In other aspects of this embodiment, Pa , at least 1,600 Pa , at least 1700 Pa, at least 1800 Pa , at a hydrogel composition exhibits a tan d of, e.g. , at most 0.1, least 1900 Pa , at least 2,000 Pa , at least 2,100 Pa , at least atmost 0.2 , at most 0.3 , at most 0.4 , at most 0.5 , at most 0.6 , 2,200 Pa, at least 2,300 Pa , at least 2,400 Pa , or at least 2500 15 atmost 0.7 , at most 0.8 , at most 0.9 , atmost 1.0 , at most 1.1 , Pa. In yet other aspects of this embodiment, a hydrogel at most 1.2 , at most 1.3 , at most 1.4 , atmost 1.5 , atmost 1.6 , composition exhibits an elastic modulus of, e.g. , at most 25 atmost 1.7 , at most 1.8 , at most 1.9 , at most 2.0 , at most 2.1 , Pa , at most 50 Pa , at most 75 Pa , atmost 100 Pa, at most 125 at most 2.2 , at most 2.3 , at most 2.4 , or at most 2.5 . In yet Pa, at most 150 Pa , at most 175 Pa , at most 200 Pa , at most other aspects of this embodiment, a hydrogel composition 250 Pa , at most 300 Pa , at most 350 Pa , at most 400 Pa, at 20 exhibits a tan dof, e.g. , about 0.1 to about 0.3 , about 0.3 to most 450 Pa, at most 500 Pa, at most 550 Pa , at most 600 about 0.5 , about 0.5 to about 0.8 , about 1.1 to about 1.4 , Pa , at most 650 Pa , at most 700 Pa , at most 750 Pa , at most about 1.4 to about 1.7 , about 0.3 to about 0.6 , about 0.1 to 800 Pa , at most 850 Pa , at most 900 Pa, at most 950 Pa , at about 0.5 , about 0.5 to about 0.9 , about 0.1 to about 0.6 , most 1,000 Pa , atmost 1,200 Pa , at most 1,300 Pa , at most about 0.1 to about 1.0 , about 0.5 to about 1.5 , about 1.0 to 1,400 Pa , at most 1,500 Pa, or at most 1,600 Pa. In still other 25 about 2.0 , or about 1.5 to about 2.5 . In some embodiments aspects of this embodiment, a hydrogel composition exhibits the tan d is less than about 0.1 , for example , is about 0.02 to an elastic modulus of, e.g. , about 25 Pa to about 150 Pa , about 0.1 , for example , about 0.05 . about 25 Pa to about 300 Pa , about 25 Pa to about 500 Pa , Aspects of the present specification provide, in part , a about 25 Pa to about 800 Pa, about 125 Pa to about 300 Pa , hydrogel composition disclosed herein having a transpar about 125 Pa to about 500 Pa, about 125 Pa to about 800 Pa, 30 ency and/ or translucency. Transparency (also called pellu about 500 Pa to about 1,600 Pa, about600 Pa to about 1,600 cidity or diaphaneity ) is the physical property of allowing Pa , about 700 Pa to about 1,600 Pa , about 800 Pa to about light to pass through a material , whereas translucency (also 1,600 Pa , about 900 Pa to about 1,600 Pa , about 1,000 Pa to called translucence or translucidity ) only allows light to pass about 1,600 Pa , about 1,100 Pa to about 1,600 Pa , about through diffusely . The opposite property is opacity . Trans 1,200 Pa to about 1,600 Pa, about 500 Pa to about 2,500 Pa , 35 parent materials are clear, while translucent ones cannot be about 1,000 Pa to about 2,500 Pa, about 1,500 Pa to about seen through clearly . The silk fibroin hydrogels disclosed 2,500 Pa , about 2,000 Pa to about 2,500 Pa , about 1,300 Pa herein may , or may not, exhibit optical properties such as to about 1,600 Pa , about 1,400 Pa to about 1,700 Pa , about transparency and translucency. In certain cases , e.g., super 1,500 Pa to about 1,800 Pa , about 1,600 Pa to about 1,900 ficial line filling, it would be an advantage to have an opaque Pa, about 1,700 Pa to about 2,000 Pa , about 1,800 Pa to 40 hydrogel. In other cases such as development of a lens or a about 2,100 Pa , about 1,900 Pa to about 2,200 Pa , about “ humor ” for filling the eye , it would be an advantage to have 2,000 Pa to about 2,300 Pa, about 2,100 Pa to about 2,400 a translucent hydrogel. These properties could be modified Pa , or about 2,200 Pa to about 2,500 Pa . by affecting the structural distribution of the hydrogelmate In another embodiment, a hydrogel composition disclosed rial. Factors used to control a hydrogel's optical properties herein exhibits a viscous modulus . In aspects of this embodi- 45 include, without limitation , polymer concentration , gel crys ment, a hydrogel composition exhibits a viscous modulus of, tallinity , and hydrogel homogeneity . e.g., about 10 Pa , about 20 Pa , about 30 Pa, about 40 Pa , When light encounters a material, it can interact with it in about 50 Pa , about 60 Pa , about 70 Pa , about 80 Pa , about several different ways. These interactions depend on the 90 Pa , about 100 Pa , about 150 Pa , about 200 Pa , about 250 nature of the light ( its wavelength , frequency , energy , etc. ) Pa, about 300 Pa, about 350 Pa, about 400 Pa , about 450 Pa , 50 and the nature of the material. Light waves interact with an about 500 Pa , about 550 Pa , about 600 Pa , about 650 Pa , or object by some combination of reflection , and transmittance about 700 Pa . In other aspects of this embodiment, a with refraction . As such , an optically transparent material hydrogel composition exhibits a viscous modulus of, e.g. , at allows much of the light that falls on it to be transmitted , most 10 Pa , at most 20 Pa , at most 30 Pa , at most 40 Pa , at with little light being reflected . Materials which do not allow most 50 Pa, at most 60 Pa , at most 70 Pa , at most 80 Pa , at 55 the transmission of light are called optically opaque or most 90 Pa , at most 100 Pa , at most 150 Pa , at most 200 Pa , simply opaque . at most 250 Pa , atmost 300 Pa , atmost 350 Pa , at most 400 In an embodiment, a hydrogel composition disclosed Pa , atmost 450 Pa , at most 500 Pa , at most 550 Pa , at most herein is optically transparent. In aspects of this embodi 600 Pa , at most 650 Pa , or at most 700 Pa . In yet other ment , a hydrogel composition transmits, e.g., about 75 % of aspects of this embodiment, a hydrogel composition exhibits 60 the light, about 80 % of the light, about 85 % of the light, a viscousmodulus of, e.g., about 10 Pa to about 30 Pa, about about 90 % of the light, about 95 % of the light, or about 10 Pa to about 50 Pa, about 10 Pa to about 100 Pa, about 10 100 % of the light. In other aspects of this embodiment, a Pa to about 150 Pa , about 70 Pa to about 100 Pa , about 50 hydrogel composition transmits , e.g., at least 75 % of the Pa to about 350 Pa , about 150 Pa to about 450 Pa , about 250 light, at least 80 % of the light, at least 85 % of the light, at Pa to about 550 Pa, about 350 Pa to about 700 Pa, about 50 65 least 90 % of the light, or at least 95 % of the light. In yet Pa to about 150 Pa , about 100 Pa to about 200 Pa , about 150 other aspects of this embodiment, a hydrogel composition Pa to about 250 Pa , about 200 Pa to about 300 Pa , about 250 transmits , e.g., about 75 % to about 100 % of the light, about US 10,722,444 B2 21 22 80 % to about 100 % of the light, about 85 % to about 100 % region of an individual using an injection device with a fine of the light, about 90 % to about 100 % of the light, or about needle . As used herein , the term “ thin needle ” refers to a 95 % to about 100 % of the light. needle that is 27 gauge or smaller . In another embodiment, a hydrogel composition disclosed In aspect of this embodiment , a hydrogel composition herein is optically opaque. In aspects of this embodiment, a 5 disclosed herein is injectable through a fine needle . In other hydrogel composition transmits , e.g., about 5 % of the light, aspects of this embodiment, a hydrogel composition dis about 10 % of the light, about 15 % of the light, about 20 % closed herein is injectable through a needle of , e.g. , about 27 of the light, about 25 % of the light, about 30 % of the light, gauge , about 30 gauge, or about 32 gauge . In yet other about 35 % of the light, about 40 % of the light, about 45 % aspects of this embodiment , a hydrogel composition dis of the light, about 50 % of the light , about 55 % of the light, 10 closed herein is injectable through a needle of, e.g., 22 gauge about 60 % of the light, about65 % of the light, or about 70 % or smaller, 27 gauge or smaller, 30 gauge or smaller, or 32 of the light. In other aspects of this embodiment, a hydrogel gauge or smaller . In still other aspects of this embodiment, composition transmits , e.g. , at most 5 % of the light, at most a hydrogel composition disclosed herein is injectable 10 % of the light, at most 15 % of the light, at most 20 % of through a needle of, e.g., about 22 gauge to about 35 gauge , the light , at most 25 % of the light , at most 30 % of the light, 15 22 gauge to about 34 gauge , 22 gauge to about 33 gauge , 22 at most 35 % of the light, at most 40 % of the light, at most gauge to about 32 gauge, about 22 gauge to about 27 gauge , 45 % of the light, at most 50 % of the light, at most 55 % of or about 27 gauge to about 32 gauge . the light, at most 60 % of the light, at most 65 % of the light, In aspects of this embodiment, a hydrogel composition at most 70 % of the light, or atmost 75 % of the light. In other disclosed herein can be injected with an extrusion force of aspects of this embodiment, a hydrogel composition trans- 20 about 60 N , about 55 N , about 50 N , about 45 N , about 40 mits , e.g., about 5 % to about 15 % , about 5 % to about 20 % , N , about 35 N , about 30 N , about 25 N , about 20 N , or about about 5 % to about 25 % , about 5 % to about 30 % , about 5 % 15 N at speeds of 100 mm /min . In other aspects of this to about 35 % , about 5 % to about 40 % , about 5 % to about embodiment, a hydrogel composition disclosed herein can 45 % , about 5 % to about 50 % , about 5 % to about 55 % , about be injected through a 27 gauge needle with an extrusion 5 % to about 60 % , about 5 % to about 65 % , about 5 % to about 25 force of about 60 N or less , about 55 N or less, about 50 N 70 % , about 5 % to about 75 % , about 15 % to about 20 % , or less , about 45 N or less , about 40 N or less, about 35 N about 15 % to about 25 % , about 15 % to about 30 % , about or less , about 30 N or less, about 25 N or less, about 20 N 15 % to about 35 % , about 15 % to about 40 % , about 15 % to or less , about 15 N or less , about 10 N or less , or about 5 N about 45 % , about 15 % to about 50 % , about 15 % to about or less . In yet other aspects of this embodiment, a hydrogel 55 % , about 15 % to about 60 % , about 15 % to about 65 % , 30 composition disclosed herein can hp injected through a 30 about 15 % to about 70 % , about 15 % to about 75 % , about gauge needle with an extrusion force of about 60 N or less, 25 % to about 35 % , about 25 % to about 40 % , about 25 % to about 55 N or less , about 50 N or less, about 45 N or less, about 45 % , about 25 % to about 50 % , about 25 % about about 40 N or less , about 35 N or less, about 30 N or less , 55 % , about 25 % to about 60 % , about 25 % to about 65 % , about 25 N or less , about 20 N or less, about 15 N or less , about 25 % to about 70 % , or about 25 % to about 75 % , of the 35 about 10 N or less , or about 5 N or less . In still other aspects light. of this embodiment, a hydrogel composition disclosed In an embodiment, a hydrogel composition disclosed herein can be injected through a 32 gauge needle with an herein is optically translucent. In aspects of this embodi extrusion force of about 60 N or less, about 55 N or less, ment, a hydrogel composition diffusely transmits , e.g., about about 50 N or less, about 45 N or less, about 40 N or less , 75 % of the light, about 80 % of the light , about 85 % of the 40 about 35 N or less, about 30 N or less , about 25 N or less , light, about 90 % of the light, about 95 % of the light, or about about 20 N or less, about 15 N or less , about 10 N or less , 100 % of the light . In other aspects of this embodiment, a or about 5 N or less . hydrogel composition diffusely transmits , e.g. , at least 75 % Aspects of the present specification provide, in part , a of the light, at least 80 % of the light, at least 85 % of the light, hydrogel composition disclosed herein that exhibits cohe at least 90 % of the light, or at least 95 % of the light. In yet 45 sivity . Cohesivity , also referred to as internal adhesion , or other aspects of this embodiment, a hydrogel composition cohesive force , is a macroscopic property of a material, diffusely transmits , e.g., about 75 % to about 100 % of the caused by the intermolecular attraction forces between HA light, about 80 % to about 100 % of the light, about 85 % to chains within the material that acts to make the HA domains / about 100 % of the light, about 90 % to about 100 % of the particles adhere together more or less . Cohesivity is mea light, or about 95 % to about 100 % of the light. 50 sured through resistance to linear compression and A hydrogel composition disclosed herein may be further expressed in terms of grams- force ( gmf) . Cohesivity is processed by pulverizing the hydrogel into particles and affected by , among other factors, the HA concentration , the optionally mixed with a carrier phase such as, e.g., water or molecular weight ratio of the initial free glycosaminoglycan a saline solution to form an injectable or topical substance polymer , the amount of residual free glycosaminoglycan like a solution , oil, lotion , gel , ointment , cream , slurry , salve , 55 polymers following crosslinking , and the pH of the hydrogel or paste . As such , the disclosed hydrogel compositionsmay composition . A composition should be sufficiently cohesive be monophasic or multiphasic compositions. A hydrogel as to remain localized to a site of administration . Addition may be milled to a particle size from about 10 um to about ally , in certain applications, a sufficient cohesivity is impor 1000 um in diameter , such as about 15 um to about 30 um , tant for a composition to retain its shape under linear about 50 um to about 75 um , about 100 um to about 150 um , 60 compression (= flattening ), and thus functionality, in the about 200 um to about 300 um , about 450 um to about 550 event ofmechanical load cycling. As such , in one embodi um , about 600 um to about 700 um , about 750 um to about ment, a hydrogel composition disclosed herein exhibits 850 um , or about 900 um to about 1,000 um . cohesivity , on par with water. In yet another embodiment, a Aspects of the present specification provide, in part, a hydrogel composition disclosed herein exhibits sufficient composition disclosed herein is injectable . As used herein , 65 cohesivity to remain localized to a site of administration . In the term “ injectable ” refers to a material having the prop still another embodiment, a hydrogel composition disclosed erties necessary to administer the composition into a skin herein exhibits sufficient cohesivity to retain its shape . US 10,722,444 B2 23 24 Specifically , a force of 20 gmf ( 0.1962 N ) or more 350 mOsm / L , about 250 mOsm / L to about 325 mOsm / L , indicates a cohesive material in the sense of the present about 275 mOsm / L to about 300 mOsm /L , or about 285 invention . Gels with lower compression force values are mOsm / L to about 290 mOsm / L . generally not considered cohesive in the context of the Aspects of the present specification provide, in part , a present invention . Preferably , the compression force mea 5 hydrogel composition disclosed herein that exhibits a physi sured as outlined above is at least 25 , 30 or 40 gmf. Most ologically -acceptable osmolality . As used herein , the term preferably , the compression force measured as outlined " osmolality ” refers to the concentration of osmotically above is at least 50 or 60 gmf. The precision of this active solutes per kilo of solvent in the body . As used herein , measurement is in the order of 25 gmf. This is most the term " a physiologically -acceptable osmolality ” refers to important for a dermal filler as the cohesivity as defined 10 an osmolality in accord with , or characteristic of, the normal above will contribute to the lift capacity ( clinically called the functioning of a living organism . As such , administration of volumizing/ bulking effect ) provided by the gel clinically , a hydrogel composition disclosed herein exhibits an osmo along with its elastic modulus G '. While cohesive gels can lality that has substantially no long term or permanent show a good volumizing effect , non -cohesive or weakly detrimental effect when administered to a mammal. Osmo cohesive materials with a similar elastic modulus exhibits 15 lality is expressed in terms of osmoles of osmotically active lower lift capacity due to the non -cohesive gel material solute per kilogram of solvent (osmol /kg or Osm /kg ) and is spreading more than a more cohesive material when sub equal to the sum of the molalities of all the solutes present mitted to vertical compression . in that solution . The osmolality of a solution can be mea Aspects of the present specification provide , in part, a sured using an osmometer. The most commonly used instru hydrogel composition disclosed herein that exhibits a physi- 20 ment in modern laboratories is a freezing point depression ologically -acceptable osmolarity . As used herein , the term osmometer . This instruments measure the change in freezing " osmolarity ” refers to the concentration of osmotically point that occurs in a solution with increasing osmolality active solutes in solution . As used herein , the term “ a ( freezing point depression osmometer) or the change in physiologically -acceptable osmolarity ” refers to an osmo vapor pressure that occurs in a solution with increasing larity in accord with , or characteristic of, the normal func- 25 osmolality (vapor pressure depression osmometer ) . tioning of a living organism . As such , administration of a In an embodiment, a hydrogel composition disclosed hydrogel composition as disclosed herein exhibits an osmo herein exhibits a physiologically -acceptable osmolality . In larity that has substantially no long term or permanent aspects ofthis embodiment, a hydrogel composition exhibits detrimental effect when administered to a mammal. Osmo an osmolality of, e.g., about 100 mOsm /kg , about 150 larity is expressed in termsof osmoles of osmotically active 30 mOsm /kg , about 200 mOsm /kg , about 250 mOsm /kg , about solute per liter of solvent (Osmol /L or Osm / L ) . Osmolarity 300 mOsm /kg , about 350 mOsm /kg , about 400 mOsm /kg , is distinct from molarity because it measures moles of about 450 mOsm /kg , or about 500 mOsm /kg . In other osmotically active solute particles rather than moles of aspects of this embodiment, a hydrogel composition exhibits solute. The distinction arises because some compounds can an osmolality of, e.g., at least 100 mOsm /kg , at least 150 dissociate in solution , whereas others cannot . The osmolar- 35 mOsm /kg , at least 200 mOsm /kg , at least 250 mOsm /kg , at ity of a solution can be calculated from the following least 300 mOsm /kg , at least 350 mOsm /kg , at least 400 expression: Osmol/ L = E Q ; n ; C ;, where y is the osmotic mOsm /kg , at least 450 mOsm /kg , or at least 500 mOsm /kg . coefficient, which accounts for the degree of non - ideality of In yet other aspects of this embodiment, a hydrogel com the solution ; n is the number of particles (e.g. ions ) into position exhibits an osmolality of , e.g. , at most 100 mOsm / which a molecule dissociates; and C is the molar concen- 40 kg, at most 150 mOsm /kg , at most 200 mOsm /kg , at most tration of the solute ; and i is the index representing the 250 mOsm /kg , at most 300 mOsm /kg , at most 350 mOsm / identity of a particular solute. The osmolarity of a hydrogel kg , at most 400 mOsm /kg , at most 450 mOsm /kg , or at most composition disclosed herein can be measured using a 500 mOsm /kg . In still other aspects of this embodiment, a conventional method that measures solutions . hydrogel composition exhibits an osmolality of, e.g., about In an embodiment, a hydrogel composition disclosed 45 100 mOsm /kg to about 500 mOsm /kg , about 200 mOsm /kg herein exhibits a physiologically - acceptable osmolarity . In to about 500 mOsm /kg , about 200 mOsm /kg to about 400 aspects of this embodiment, a hydrogel composition exhibits mOsm /kg , about 300 mOsm /kg to about 400 mOsm /kg , an osmolarity of, e.g. , about 100 mOsm /L , about 150 about 270 mOsm /kg to about 390 mOsm /kg , about 225 mOsm / L , about 200 mOsm / L , about 250 mOsm /L , about mOsm /kg to about 350 mOsm /kg , about 250 mOsm /kg to 300 mOsm / L , about 350 mOsm / L , about 400 mOsm / L , 50 about 325 mOsm /kg , about 275 mOsm /kg to about 300 about 450 mOsm / L , or about 500 mOsm / L . In other aspects mOsm /kg , or about 285 mOsm /kg to about 290 mOsm /kg . of this embodiment, a hydrogel composition exhibits an Aspects of the present specification provide, in part , a osmolarity of, e.g. , at least 100 mOsm / L , at least 150 hydrogel composition disclosed herein that exhibits substan mOsm / L , at least 200 mOsm / L , at least 250 mOsm / L , at tial stability . As used herein , the term “ stability ” or “ stable ” least 300 mOsm / L , at least 350 mOsm /L , at least 400 55 when referring to a hydrogel composition disclosed herein mOsm /L , at least 450 mOsm /L , or at least 500 mOsm /L . In refers to a composition that is not prone to degrading, yet other aspects of this embodiment , a hydrogel composi decomposing, or breaking down to any substantial or sig tion exhibits an osmolarity of, e.g. , at most 100 mOsm / L , at nificant degree while stored before administration to an most 150 mOsm / L , at most 200 mOsm / L , at most 250 individual. As used herein , the term “ substantial heat sta mOsm /L , at most 300 mOsm /L , at most 350 mOsm /L , at 60 bility ”, “ substantially heat stable ” , “ autoclave stable ” , or most 400 mOsm / L , at most 450 mOsm / L , or at most 500 " steam sterilization stable ” refers to a hydrogel composition mOsm / L . In still other aspects of this embodiment, a hydro disclosed herein that is substantially stable when subjected gel composition exhibits an osmolarity of, e.g., about 100 to a heat treatment as disclosed herein . mOsm / L to about 500 mOsm / L , about 200 mOsm /L to about Stability of a hydrogel composition disclosed herein can 500 mOsm / L , about 200 mOsm / L to about 400 mOsm /L , 65 be determined by subjecting a hydrogel composition to a about 300 mOsm / L to about 400 mOsm /L , about 270 heat treatment , such as, e.g. , steam sterilization at normal mOsm / L to about 390 mOsm / L , about 225 mOsm / L to about pressure or under pressure ( e.g. , autoclaving ). Preferably the US 10,722,444 B2 25 26 heat treatment is carried out at a temperature of at least about the specified additives before and after the 45 ° C. heat 100 ° C. for between about one minute and about 15 minutes . treatment) ; and 3 ) by determining the change in rheological Substantial stability of a hydrogel composition disclosed properties of a hydrogel composition disclosed herein after herein can be evaluated 1 ) by determining the change in the sterilization , where the change in tan d 1 Hz of less than 0.1 extrusion force (AF ) of a hydrogel composition disclosed 5 is indicative of a substantially stable hydrogel composition herein after sterilization ,where the change in extrusion force as measured by A Tan d 1 Hz = ( tan 8 1 Hz of gel formulation less 2N is indicative of a substantially stable hydrogel with the specified additives before and after the 45 ° C. heat composition as measured by ( the extrusion force of a treatment) minus ( tan d 1 Hz of gel formulation without the hydrogel composition with the specified additives) minus specified additives before and after the 45 ° C. heat treat ( the extrusion force of the a hydrogel composition without 10 ment ) . As such , a long term stability of a hydrogel compo the added additives ); and / or 2 ) by determining the change in sition disclosed herein is evaluated by retention of one or rheological properties of a hydrogel composition disclosed more of the following characteristics after the 45 ° C. heat herein after sterilization , where the change in tan 8 1 Hz of treatment: clarity ( transparency and translucency ), homoge less than 0.1 is indicative of a substantially stable hydrogel neousness , and cohesiveness . composition as measured by A Tan 8 1 Hz = (tan 8 1 Hz of gel 15 In aspects of this embodiment, when A Tan 8 1 Hz formulation with additives ) minus ( tan 1 Hz of gel becomes negative over sterilization , the formulation with the formulation without additives ) . As such , a substantially antioxidant is more stable over sterilization . stable hydrogel composition disclosed herein retains one or In aspects of this embodiment, a hydrogel composition is more of the following characteristics after sterilization : substantially stable at room temperature for, e.g., about 3 homogeneousness , extrusion force, cohesiveness , hyaluro- 20 months, about 6 months, about 9 months, about 12 months, nan concentration , agent( s ) concentration , osmolarity , pH , about 15 months , about 18 months, about 21 months , about or other rheological characteristics such as tan d . 24 months, about 27 months , about 30 months, about 33 In an aspect of this embodiment, when the change in months, or about 36 months . In other aspects of this embodi rheological properties becomes negative (ie . tan d lower ment, a hydrogel composition is substantially stable at room after sterilization with the additive) , the formulation is said 25 temperature for, e.g., at least 3 months , at least 6 months, at to be more stable over sterilization due to the antioxidant . least 9 months, at least 12 months, at least 15 months , at least In an embodiment, a hydrogel composition comprising a 18 months, at least 21 months, at least 24 months, at least 27 glycosaminoglycan polymer and the at least one agent months, at least 30 months, at least 33 months, or at least 36 disclosed herein is processed using a heat treatment that months. In other aspects of this embodiment, a hydrogel maintains the desired hydrogel properties disclosed herein . 30 composition is substantially stable at room temperature for, In aspects of this embodiment, a hydrogel composition e.g., about 3 months to about 12 months, about 3 months to comprising a glycosaminoglycan polymer and the at least about 18 months, about 3 months to about 24 months , about one agent disclosed herein is processed using a heat treat 3 months to about 30 months , about 3 months to about 36 ment of, e.g. , about 100 ° C., about 105 ° C., about 110 ° C., months , about 6 months to about 12 months, about 6 months about 115 ° C., about 120 ° C., about 125 ° C., or about 130 ° 35 to about 18 months, about 6 months to about 24 months , C. In other aspects of this embodiment, a hydrogel compo about 6 months to about 30 months, about 6 months to about sition comprising a glycosaminoglycan polymer and the at 36 months, about 9 months to about 12 months, about 9 least one agent disclosed herein is processed using a heat months to about 18 months, about 9 months to about 24 treatment of, e.g., at least 100 ° C., at least 105 ° C., at least months, about 9 months to about 30 months, about 9 months 110 ° C., at least 115º C., at least 120 ° C., at least 125 ° C., 40 to about 36 months, about 12 months to about 18 months , or at least 130 ° C. In yet other aspects of this embodiment, about 12 months to about 24 months , about 12 months to a hydrogel composition comprising a glycosaminoglycan about 30 months , about 12 months to about 36 months, about polymer and the at least one agent disclosed herein is 18 months to about 24 months, about 18 months to about 30 processed using a heat treatment of, e.g. , about 100 ° C. to months, or about 18 months to about 36 months. about 120 ° C., about 100 ° C. to about 125 ° C., about 100 ° 45 In aspects of this embodiment , the resistance of formu C. to about 130 ° C., about 100 ° C. to about 135 ° C., about lations to degradation by free radicals can be assessed to 110 ° C. to about 120 ° C., about 110 ° C. to about 125 ° C., characterize the effect of the antioxidant. Degradation by about 110 ° C. to about 130 ° C., about 110 ° C. to about 135 ° free radicals was simulated on a rheometer ( Haake Rheo C., about 120 ° C. to about 125 ° C., about 120 ° C. to about stress 600 ) by addition of 1/7 ratio of H202 30 % on the 130 ° C., about 120 ° C. to about 135 ° C., about 125 ° C. to 50 surface of a spread gel measured with a controlled stress about 130 ° C., or about 125 ° C. to about 135º C. rheometer according to the following method : frequency of Long term stability of a hydrogel composition disclosed 1 Hz with 0.8 % controlled strain , during 3600 seconds at 35 ° herein can be determined by subjecting a hydrogel compo C. The complex viscosity or elastic modulus decrease is sition to a heat treatment, such as, e.g., storage in an about followed versus time and the value of the slope to charac 45 ° C. environment for about 60 days . Long term stability of 55 terizes the speed of free radical degradation . The time it a hydrogel composition disclosed herein can be evaluated 1 ) takes for complex viscosity or elastic modulus to decrease by assessing the clarity and color of a hydrogel composition by 70 % is called T30 . Comparing T30 with and without the after the 45 ° C. heat treatment, with a clear and uncolored antioxidant is a measurement of potential duration . hydrogel composition being indicative of a substantially Aspects of the present specification provide, in part, a stable hydrogel composition ; 2) by determining the change 60 method of treating a soft tissue condition of an individual by in the extrusion force (AF ) of a hydrogel composition administering a hydrogel composition disclosed herein . As disclosed herein after the 45 ° C. heat treatment, where the used herein , the term “ treating ,” refers to reducing or elimi change in extrusion force less 2N is indicative of a substan nating in an individual a cosmetic or clinical symptom of a tially stable hydrogel composition as measured by the soft tissue condition characterized by a soft tissue imper extrusion force of a hydrogel composition with the specified 65 fection , defect , disease , and/ or disorder ; or delaying or additives before and after the 45 ° C. heat treatment ) minus preventing in an individual the onset of a cosmetic or clinical ( the extrusion force of the a hydrogel composition without symptom of a condition characterized by a soft tissue US 10,722,444 B2 27 28 imperfection , defect , disease , and /or disorder . For example , tered as small multiple droplets into the epidermis , dermo the term “ treating ” can mean reducing a symptom of a epidermal junction , and /or the dermis . condition characterized by a soft tissue defect , disease , The amount of a hydrogel composition used with any of and /or disorder by, e.g. , at least 20 % , at least 30 % , at least the methods as disclosed herein will typically be determined 40 % , at least 50 % , at least 60 % , at least 70 % , at least 80 % , 5 based on the alteration and /or improvement desired , the at least 90 % or at least 100 % . The effectiveness of a reduction and / or elimination of a soft tissue condition symp hydrogel composition disclosed herein in treating a condi tom desired , the clinical and /or cosmetic effect desired by tion characterized by a soft tissue defect, disease , and /or the individual and /or physician , and the body part or region disorder can be determined by observing one or more being treated . The effectiveness of composition administra cosmetic , clinical symptoms, and / or physiological indicators 10 tion may be manifested by one or more of the following associated with the condition . An improvement in a soft clinical and /or cosmetic measures : altered and / or improved tissue defect, disease, and /or disorder also can be indicated soft tissue shape , altered and /or improved soft tissue size , by a reduced need for a concurrent therapy. Those of skill in altered and /or improved soft tissue contour, altered and /or the art will know the appropriate symptoms or indicators improved tissue function , tissue ingrowth support and /or associated with specific soft tissue defect, disease , and / or 15 new collagen deposition , sustained engraftment of compo disorder and will know how to determine if an individual is sition , improved patient satisfaction and/ or quality of life , a candidate for treatment with a compound or composition and decreased use of implantable foreign material . disclosed herein . For example , for breast augmentation procedures, effec A hydrogel composition is administered to an individual. tiveness of the compositions and methods may be mani An individual is typically a human being of any age, gender 20 fested by one or more of the following clinical and / or or race . Typically , any individual who is a candidate for a cosmetic measures: increased breast size, altered breast conventional procedure to treat a soft tissue condition is a shape, altered breast contour, sustained engraftment, reduc candidate for a method disclosed herein . Although a subject tion in the risk of capsular contraction , decreased rate of experiencing the signs of aging skin is an adult, subjects liponecrotic cyst formation , improved patient satisfaction experiencing premature aging or other skin conditions suit- 25 and / or quality of life , and decreased use of breast implant . able for treatment ( for example , a scar ) can also be treated As another example , effectiveness of the compositions with a hydrogel composition disclosed herein . In addition , and methods in treating a facial soft tissue may be mani the presently disclosed hydrogel compositions and methods fested by one or more of the following clinical and /or may apply to individuals seeking a small/ moderate enlarge cosmetic measures: increased size , shape , and /or contour of ment, shape change or contour alteration of a body part or 30 facial feature like increased size , shape, and /or contour of region , which may not be technically possible or aestheti lip , cheek or eye region ; altered size , shape , and /or contour cally acceptable with existing soft tissue implant technology . of facial feature like altered size , shape, and /or contour of Pre -operative evaluation typically includes routine history lip , cheek or eye region shape ; reduction or elimination of a and physical examination in addition to thorough informed wrinkle , fold or line in the skin ; resistance to a wrinkle , fold consent disclosing all relevant risks and benefits of the 35 or line in the skin ; rehydration of the skin ; increased procedure. elasticity to the skin ; reduction or elimination of skin The hydrogel composition and methods disclosed herein roughness ; increased and /or improved skin tautness ; reduc are useful in treating a soft tissue condition . A soft tissue tion or elimination of stretch lines or marks ; increased condition includes , without limitation , a soft tissue imper and / or improved skin tone, shine , brightness and / or radi fection , defect, disease , and /or disorder. Non -limiting 40 ance; increased and /or improved skin color, reduction or examples of a soft tissue condition include breast imperfec elimination of skin paleness; sustained engraftment of com tion , defect, disease and /or disorder , such as, e.g., a breast position ; decreased side effects ; improved patient satisfac augmentation , a breast reconstruction , mastopexy, micro tion and /or quality of life. mastia , thoracic hypoplasia , Poland's syndrome, defects due As yet another example , for urinary incontinence proce to implant complications like capsular contraction and /or 45 dures , effectiveness of the compositions and methods for rupture; a facial imperfection , defect, disease or disorder , sphincter support may be manifested by one or more of the such as, e.g. , a facial augmentation , a facial reconstruction , following clinical measures : decreased frequency of incon a mesotherapy, Parry -Romberg syndrome, lupus erythema tinence , sustained engraftment, improved patient satisfac tosus profundus, dermal divots , scars , sunken cheeks, thin tion and / or quality of life , and decreased use of implantable lips , nasal imperfections or defects , retro -orbital imperfec- 50 foreign filler . tions or defects , a facial fold , line and /or wrinkle like a In aspects of this embodiment, the amount of a hydrogel glabellar line , a nasolabial line , a perioral line , and / or a composition administered is , e.g., about 0.01 g , about 0.05 marionette line , and /or other contour deformities or imper g , about 0.1 g , about 0.5 g , about 1 g , about 5 g, about 10 fections of the face ; a neck imperfection , defect, disease or g , about 20 g about 30 g about 40 g , about 50 g , about 60 disorder ; a skin imperfection , defect, disease and / or disor- 55 g , about 70 g , about 80 g , about 90 g , about 100 g , about 150 der ; other soft tissue imperfections, defects , diseases and / or g , or about 200 g . In other aspects of this embodiment, the disorders , such as , e.g., an augmentation or a reconstruction amount of a hydrogel composition administered is , e.g. , of the upper arm , lower arm , hand , shoulder, back , torso about 0.01 g to about 0.1 g , about 0.1 g to about 1 g , about including abdomen , buttocks, upper leg , lower leg including 1 g to about 10 g, about 10 g to about 100 g , or about 50 g calves, foot including plantar fat pad , eye, genitals , or other 60 to about 200 g . In yet other aspects of this embodiment, the body part, region or area, or a disease or disorder affecting amount of a hydrogel composition administered is , e.g., these body parts , regions or areas; urinary incontinence , about 0.01 mL , about 0.05 mL , about 0.1 mL, about 0.5 mL , fecal incontinence , other forms of incontinence ; and gas about 1 mL , about 5 mL , about 10 mL , about 20 mL , about troesophageal reflux disease (GERD ) . As used herein , the 30 mL , about 40 mL , about 50 mL , about 60 mL , about 70 term “ mesotherapy ” refers to a non - surgical cosmetic treat- 65 g , about 80 mL ,9 about 90 mL , about 100 mL , about 150 mL , ment technique of the skin involving intra - epidermal , intra or about 200 mL . In other aspects of this embodiment, the dermal, and /or subcutaneous injection of an agent adminis amount of a hydrogel composition administered is , e.g. , US 10,722,444 B2 29 30 about 0.01 mL to about 0.1 mL , about 0.1 mL to about 1 mL , For example , a hydrogel composition disclosed herein can about 1 mL to about 10 mL , about 10 mL to about 100 mL , be injected utilizing needles with a diameter of about 0.26 or about 50 mL to about 200 mL . mm to about 0.4 mm and a length ranging from about 4 mm The duration of treatment will typically be determined to about 14 mm . Alternately , the needles can be 21 to 32 G based on the cosmetic and /or clinical effect desired by the 5 and have a length of about 4 mm to about 70 mm . Preferably , individual and /or physician and the body part or region the needle is a single -use needle . The needle can be com being treated . In aspects of this embodiment , administration bined with a syringe , catheter , and / or a pistol . of a hydrogel composition disclosed herein can treat a soft In addition , a composition disclosed herein can be admin tissue condition for , e.g., about 6 months, about 7 months, istered once, or over a plurality of times. Ultimately , the about 8 months , about 9 months , about 10 months, about 11 10 timing used will follow quality care standards. For example , months, about 12 months, about 13 months, about 14 a hydrogel composition disclosed herein can be adminis months, about 15 months, about 18 months, or about 24 tered once or over several sessions with the sessions spaced months. In other aspects of this embodiment , administration apart by a few days, or weeks. For instance , an individual of a hydrogel composition disclosed herein can treat a soft can be administered a hydrogel composition disclosed tissue condition for , e.g. , at least 6 months, at least 7 months, 15 herein every 1, 2 , 3, 4 , 5 , 6 , or 7 days or every 1 , 2 , 3 , or 4 at least 8 months, at least 9 months, at least 10 months , at weeks . The administration a hydrogel composition disclosed least 11 months, at least 12 months, at least 13 months, at herein to an individual can be on a monthly or bi- monthly least 14 months, at least 15 months, at least 18 months, or basis or administered every 3 , 6 , 9 , or 12 months. at least 24 months. In yet aspects of this embodiment, For a breast soft tissue replacement procedure , the route administration of a hydrogel composition disclosed herein 20 of administration may include axillary , periareolar , and /or can treat a soft tissue condition for, e.g., about 6 months to inframammary routes . Alternatively or in addition , a com about 12 months, about 6 months to about 15 months, about position may be delivered through a transaxillary endo 6 months to about 18 months , about 6 months to about 21 scopic subpectoral approach . For a facial soft tissue replace months, about 6 months to about 24 months, about 9 months ment procedure , the route of administration can be frontal , to about 12 months, about 9 months to about 15 months , 25 temporal, zygomatic , periocular ,mandibula , perioral or chin about 9 months to about 18 months, about 9 months to about routes. In urinary incontinence procedures, the route of 21 months , about 6 months to about 24 months , about 12 administration may include transurethral or periurethral months to about 15 months , about 12 months to about 18 routes . Alternatively or in addition , administration may be months, about 12 months to about 21 months, about 12 delivered via an antegrade route . The routes discussed herein months to about 24 months, about 15 months to about 18 30 do not exclude the use of multiple routes to achieve the months, about 15 months to about 21 months, about 15 desired clinical effect . months to about 24 months , about 18 months to about 21 Aspects of the present specification provide , in party a ths, about 18 months to about 24 months, or about 21 dermal region . As used herein , the term “ dermal region ” months to about 24 months. refers to the region of skin comprising the epidermal- dermal Aspects of the present specification provide, in part, 35 junction and the dermis including the superficial dermis administering a hydrogel composition disclosed herein . As (papillary region ) and the deep dermis ( reticular region ). The used herein , the term “ administering” means any delivery skin is composed of three primary layers : the epidermis , mechanism that provides a composition disclosed herein to which provides waterproofing and serves as a barrier to an individual that potentially results in a clinically , thera infection ; the dermis, which serves as a location for the peutically , or experimentally beneficial result. The actual 40 appendages of skin ; and the hypodermis ( subcutaneous delivery mechanism used to administer a composition to an adipose layer ) . The epidermis contains no blood vessels , and individual can be determined by a person of ordinary skill in is nourished by diffusion from the dermis . The main type of the art by taking into account factors , including , without cells which make up the epidermis are keratinocytes , mel limitation , the type of skin condition , the location of the skin anocytes , Langerhans cells and Merkels cells . condition , the cause of the skin condition , the severity of the 45 The dermis is the layer of skin beneath the epidermis that skin condition , the degree of relief desired , the duration of consists of connective tissue and cushions the body from relief desired , the particular composition used , the rate of stress and strain . The dermis is tightly connected to the excretion of the particular composition used , the pharmaco epidermis by a basement membrane. It also harbors many dynamics of the particular composition used , the nature of Mechanoreceptor/ nerve endings that provide the sense of the other compounds included in the particular composition 50 touch and heat . It contains the hair follicles , sweat glands, used , the particular route of administration , the particular sebaceous glands, apocrine glands, lymphatic vessels and characteristics, history and risk factors of the individual, blood vessels . The blood vessels in the dermis provide such as , e.g. , age , weight, general health and the like, or any nourishment and waste removal from its own cells as well as combination thereof. In an aspect of this embodiment, a from the Stratum basale of the epidermis . The dermis is composition disclosed herein is administered to a skin 55 structurally divided into two areas : a superficial area adja region of an individual by injection . cent to the epidermis , called the papillary region , and a deep The route of administration of a hydrogel composition to thicker area known as the reticular region . an individual patient will typically be determined based on The papillary region is composed of loose areolar con the cosmetic and /or clinical effect desired by the individual nective tissue . It is named for its fingerlike projections called and / or physician and the body part or region being treated . 60 papillae that extend toward the epidermis . The papillae A composition disclosed herein may be administered by any provide the dermis with a " bumpy " surface that interdigi means known to persons of ordinary skill in the art includ tates with the epidermis , strengthening the connection ing, without limitation , syringe with needle , a pistol ( for between the two layers of skin . The reticular region lies deep example , a hydropneumatic- compression pistol) , catheter, in the papillary region and is usually much thicker . It is topically , or by direct surgical implantation . The hydrogel 65 composed of dense irregular connective tissue , and receives composition disclosed herein can be administered into a skin its name from the dense concentration of collagenous , region such as , e.g. , a dermal region or a hypodermal region . elastic , and reticular fibers that weave throughout it. These US 10,722,444 B2 31 32 protein fibers give the dermis its properties of strength , administering to an individual suffering from a skin stretch extensibility , and elasticity . Also located within the reticular line or mark a hydrogel composition disclosed herein , region are the roots of the hair , sebaceous glands, sweat wherein the administration of the composition reduces or glands , receptors , nails , and blood vessels . Tattoo ink is held eliminates the skin stretch line or mark , thereby treating a in the dermis. Stretch marks from pregnancy are also located 5 mentskin ,stretch a method line of or treatingmark . Inskin another paleness aspect comprises of this embodithe step in the dermis . of administering to an individual suffering from skin pale The hypodermis lies below the dermis . Its purpose is to ness a hydrogel composition disclosed herein , wherein the attach the dermal region of the skin to underlying bone and administration of the composition increases skin tone or muscle as well as supplying it with blood vessels and nerves . radiance , thereby treating skin paleness . In another aspect of It consists of loose connective tissue and elastin . The main 10 this embodiment, a method of treating skin wrinkles com cell types are fibroblasts , macrophages and adipocytes ( the prises the step of administering to an individual suffering hypodermis contains 50 % of body fat) . Fat serves as padding from skin wrinkles a hydrogel composition disclosed herein , and insulation for the body. wherein the administration of the composition reduces or In an aspect of this embodiment, a hydrogel composition eliminates skin wrinkles, thereby treating skin wrinkles. In disclosed herein is administered to a skin region of an 15 yet another aspect of this embodiment, a method of treating individual by injection into a dermal region or a hypodermal skin wrinkles comprises the step of administering to an region . In aspects of this embodiment, a hydrogel compo individual a hydrogel composition disclosed herein , wherein sition disclosed herein is administered to a dermal region of the administration of the composition makes the skin resis an individual by injection into , e.g., an epidermal- dermal tant to skin wrinkles , thereby treating skin wrinkles . junction region , a papillary region , a reticular region , or any 20 combination thereof . EXAMPLES Aspects of the present specification disclose , in part, a The following examples illustrate representative embodi method of treating a soft tissue condition of an individual , the method comprising the steps of administering a hydrogel mentslimit the now disclosed contemplated hydrogel , but compositions should not , beand construed methods ofto composition disclosed herein to a site of the soft tissue 25 soft tissue augmentation using such hydrogel compositions. condition of the individual, wherein the administration of Reminder for stability assessment with extrusion force : the composition improves the soft tissue condition , thereby AF ( F gel with additive -F gel without additive ) < 2N treating the soft tissue condition . In aspects of this embodi Reminder for stability assessment with rheology : ATan & ment, a soft tissue, condition is a breast tissue condition , a ( Tan 8 1 Hz gel with additive -Tan d 1 Hz gel without facial tissue condition , a neck condition , a skin condition , an 30 additive ) < 0.1 upper arm condition , a lower arm condition , a hand condi Example 1 tion , a shoulder condition , a back condition , a torso includ ing abdominal condition , a buttock condi an upper leg Effect of Caffeic Acid Agent on HA - Based Gel condition , a lower leg condition including calf condition , a Formulation Incorporation and Autoclaving foot condition including plantar fat pad condition , an eye 35 Stability condition , a genital condition , or a condition effecting another body part , region or area . Caffeic acid , at a concentration of 0.6 % ,( w /w ), was incor Other aspects of the present specification disclose , in part , porated into a HA -based gel matrix at 24 mg/ g containing a method of treating a skin condition comprises the step of 5 % of uncrosslinked HA . The gel formulated was auto administering to an individual suffering from a skin condi- 40 claved . The gel obtained before autoclaving was clear and tion a hydrogel composition disclosed herein , wherein the brown , the gel obtained after autoclaving was similar. administration of the composition improves the skin condi Rheological and extrusion force properties of the gel were tion , thereby treating the skin condition . In an aspect of this analyzed and determined as shown below in Table 2 : embodiment, a skin condition is a method of treating skin dehydration comprises the step of administering to an indi- 45 TABLE 2 vidual suffering from skin dehydration a hydrogel compo sition disclosed herein , wherein the administration of the Physical properties before and after autoclaving . composition rehydrates the skin , thereby treating skin dehy Before autoclaving After autoclaving dration . In another aspect of this embodiment, a method of treating a lack of skin elasticity comprises the step of 50 ATand ATan S administering to an individual suffering from a lack of skin ApH AF ( N ) ( 1 Hz) ApH AF ( N ) ( 1 Hz ) elasticity a hydrogel composition disclosed herein , wherein 0.18 0.2 -0.003 0.55 2.9 0.02 the administration of the composition increases the elasticity of the skin , thereby treating a lack of skin elasticity . In yet another aspect of this embodiment, a method of treating skin 55 Extrusion analysis showed the beginning of degradation roughness comprises the step of administering to an indi of the uncrosslinked hyaluronic acid . vidual suffering from skin roughness a hydrogel composi Example 2 tion disclosed herein , wherein the administration of the composition decreases skin roughness , thereby treating skin Effect of Caffeic Acid Agent on HA -Based Gel roughness . In still another aspect of this embodiment, a 60 Formulation Long Term Stability method of treating a lack of skin tautness comprises the step of administering to an individual suffering from a lack of The formulations prepared in Example 1 were tested for skin tautness a hydrogel composition disclosed herein , stability at 45 ° C. for 60 days and compared to a HA - based wherein the administration of the composition makes the gel matrix without any added additives. After 30 days at 45 ° skin tauter, thereby treating a lack of skin tautness . 65 C., the gel became dark brown, and physical analysis of the In a further aspect of this embodiment, a method of gel showed a degradation of the formulation as shown by the treating a skin stretch line or mark comprises the step of values contained in Table 3 below . US 10,722,444 B2 33 34 TABLE 3 Physical properties over time. 10 days at 45 ° C. 20 days at 45 ° C. 30 days at 45 ° C. ATan 8 ATan 8 ATand ??? AF ( N ) ( 1 Hz ) ApH AF (N ) ( 1 Hz) ApH AF ( N ) ( 1 Hz ) 1.05 4.3 0.0560 0.90 4.7 0.077 1.06 4.9 0.144

Based on these results, it was concluded that direct Based on the results of this example , it was determined incorporation of caffeic acid in the gel leads to the degra that the incorporation of Inoveol CAFA in a gel did not dation of the final product. degrade physical properties of the gel, as opposed to caffeic acid (Example 2 ). Example 3 15 Example 5 Effect of Inoveol CAFA Agent on HA -Based Gel Formulation Incorporation and Autoclaving Inoveol® CAFA Protects HA -Based Gel Stability 20 Formulation from Oxidative Degradation Inoveol® CAFA (supplied by Libragen ), ( INCI name: The effect of Inoveol® CAFA on HA -based gel matrix Aqua (and ) Caffeyl Glucoside ), a derivative of caffeic acid , oxidative degradation was studied . An oxidation test was at a concentration of 0.5 % ( w /w ) of commercial solution , used as it allows for a determination of the resistance of a was incorporated into a HA -based gel matrix containing a 25 HA -based gel matrix to free radicals . Degradation by free viscoelastic portion . The gel formulated was autoclaved . radicals was simulated on a rheometer (Haake Rheostress The gel obtained before autoclaving was clear and slightly 600 ) by addition of 1/7 ratio ofH20 , 30 % on the surface of yellow , the gel obtained after autoclaving was similar . a spread gel measured with a controlled stress rheometer Rheological and extrusion force properties of the gel were according to the following method : frequency of 1 Hz with analyzed and are summarized in Table 4 below . 30 0.8 % controlled strain , during 3600 seconds at 35 ° C. The value of the slope characterizes the speed of free radical TABLE 4 degradation. The results of the oxidation test are illustrated Physical properties before and after autoclaving . in FIG . 4. A comparison of antioxidant properties after 35 autoclaving for a HA - based gel matrix with 0.5 % ( w / w ) Before autoclaving After autoclaving Inoveol® CAFA ( slope: -0.0028 ) versus the same HA -based gel matrix ( slope : -0.0041 ) showed that the gel containing ApH AF ( N ) ATand ApH AF ( N ) ATan Inoveol® CAFA is more stable with respect to free radical -0.23 0.8 0.01 -0.18 1.6 0.02 degradation . T30 for the HA -based gel matrix with 0.5 % 40 ( w / w ) Inoveol® CAFA is about 5947 seconds versus 3978 seconds for the HA -based gel matrix alone . This is about a Based on the above results , it was concluded that the gel 49% improvement due to the additive . with Inoveol® CAFA was stable after autoclaving , as comparison of antioxidant properties after thermal opposed to the same gel with caffeic acid . stability at 45 ° C. for 60 days for a HA -based gel matrix with Example 4 45 0.5 % ( w /w ) Inoveol® CAFA (slope : -0.0010 ) versus the same HA -based gel matrix ( slope : -0.0023 ) showed that the Effect of Inoveol® CAFA Agent on HA -Based Gel gel containing Inoveol® CAFA is more stable with respect Formulation Long Term Stability to free radical degradation . T30 for the HA -based gel matrix with 0.5 % ( w / w ) Inoveol® CAFA is about 10198 seconds The formulation prepared in Example 3 was subjected to 50 versus 5085 seconds for the HA - based gel matrix alone . This accelerated aging or accelerated degradation in order to test is a 100 % improvement due to the additive. These results are the stability of the gel. Specifically , the gel was tested for illustrated in FIG . 5 . stability at 45 ° C. for 60 days and compared to a HA -based Example 6 gel matrix without any added additives. The results are 55 shown in Table 5 below . FIGS . 1-3 also illustrate experi Effect of Catechin Agent on HA - Based Gel mental data obtained in this experiment that demonstrate the Formulation Incorporation and Autoclaving effect of time on the physical properties of the tested gel . Stability

TABLE 5 60 Catechin , at a concentration of 0.1 % of commercial Physical properties after 60 days . solution , was incorporated into a HA -based gel matrix at 24 60 days at 45 ° C. mg/ g containing 5 % of uncrosslinked HA . The gel formu lated was autoclaved . The gel obtained before autoclaving ApH AF ( N ) A Tan 8 was clear and slightly orange, and the gel obtained after -0.18 1.9 0.03 65 autoclaving was clear with orange color. Rheological and extrusion force analysis showed a deg radation of the gel as summarized in Table 6 below . US 10,722,444 B2 35 36 TABLE 6 Example 9 Physical properties before and after autoclaving . Inoveol® EGCG Protects HA -Based Gel Before autoclaving After autoclaving 5 Formulation from Oxidative Degradation ApH AF ( N ) ATan 8 ?pH AF ( N ) ATan 8 The effect of Inoveol® EGCG on HA - based gel matrix -0.11 0 0.008 -0.35 6.4 0.162 oxidative degradation was studied . Oxidation testing was used as it allows testing of the resistance of a HA -based gel 10 matrix to free radicals . Degradation by free radicals was Example 7 simulated on a rheometer ( Haake Rheostress 600 ) by addi tion of 1/7 ratio of H2O2 30 % on the surface of a spread gel measured with a controlled stress rheometer according to the Effect of Inoveol EGCG Agent on HA - Based Gel following method : frequency of 1 Hz with 0.8 % controlled Formulation Incorporation and Autoclaving 15 strain , during 3600 seconds at 35 ° C. The value of the slope Stability to characterizes the speed of free radical degradation . The results are illustrated in FIG . 9. A comparison of antioxidant Inoveol EGCG (supplied by Libragen ), ( INCI Name: properties after autoclaving for a HA -based gel matrix with Epigallocatechin Gallatyl Glucoside ), a derivative of cat 20 0.5 % ( w / w ) Inoveol® EGCG ( slope : -0.0019 ) versus the echin , at a concentration of 0.5 % of commercial solution , same HA -based gel matrix ( slope : -0.0039 ) showed that the was incorporated into a HA -based gel matrix containing gel containing Inoveol EGCG is more stable with respect to viscoelastic portion . The gel formulated was autoclaved . free radical degradation . T30 for the HA -based gel matrix The gel obtained before autoclaving was clear and slightly with 0.5 % (w /w ) Inoveol® EGCG is about 11308 seconds yellow and the gel obtained after autoclaving was similar . 25 versus 4663 seconds for the HA -based gel matrix alone . This Rheological and extrusion force properties of the gel were is about a 142 % improvement due to the additive . analyzed and showed the autoclaving stability of the gel, as A comparison of antioxidant properties after thermal summarized in Table 7 below . stability at 45 ° C., 60 days for a HA -based gel matrix with 0.5 % ( w /w ) Inoveol® EGCG ( slope: -0.0006 ) versus the TABLE 7 30 same HA -based gel matrix (slope : -0.0022 Pa ) showed that the gel containing Inoveol® EGCG is more stable with Physical properties before and after autoclaving . respect to free radical degradation . T30 for theHA -based gel Before autoclaving After autoclaving matrix with 0.5 % ( w / w ) Inoveol® EGCG is about 25676 seconds versus 5461 seconds for the HA -based gel matrix ?pH AF (N ) ATand ApH AF ( N ) ATan d 35 alone. This is about a 370 % improvement due to the -0.17 -0.1 0 -0.26 -1.0 -0.01 additive . These results are illustrated in FIG . 10 . Based on this example , it was concluded that the gel Example 10 containing Inoveol EGCG is stable to autoclaving and 40 slightly more stable with the additive than without. Use of Dermal Filler Composition for Treating Aging Skin Example 8 45 This Example illustrates the use of a compositions of the Effect of Inoveol EGCG Agent on HA - Based Gel invention for treating aging skin . Formulation Long Term Stability A 44 -year - old woman presents with uneven texture on her right cheek resulting from a loss of collagen due to aging . The formulation prepared in Example 7 was tested for Pre -operative evaluation of the person includes routine stability at 45° C. for 60 days and compared to a HA -based 50 historyinformed and consent physical disclosing examination all relevant in addition risks and to benefits thorough of gel matrix without any added additives. The results are the procedure . The physician evaluating the individual deter summarized in Table 8 below . mines that she is a candidate for soft tissue treatment using Additional analysis is illustrated in FIGS. 6-7 . dermal filler comprising a crosslinked hyaluronic acid 55 containing Caffeyl Glucoside , a derivative of caffeic acid at TABLE 8 a concentration of 0.5 % (w /w ), and a HA concentration of 20 Physical properties after 60 days . mg/ mL . The composition is administered subcutaneously 60 days at 45 ° C. and under superficial musculature of the affected regions once a week for three weeks, about 3.0 mL to about 4.0 mL ApH AF ( N ) A Tan 8 60 of composition into the affected cheek region . The indi -0.6 -0.05 vidual is then monitored for approximately 7 days . The a . -0.9 physician evaluates the facial regions and determines that the treatment was successful. Both the woman and her Based , at least in part, on the results of this Example , it physician are satisfied with the results of the procedure was concluded that the incorporation of Inoveol® EGCG in 65 because the texture on her face has improved . Approxi gel had no negative impact on the physical properties of the mately one month after the procedure , the woman indicates gel . (See FIG . 8 ) that her quality of life has improved . US 10,722,444 B2 37 38 Example 11 for example , the compositions and methodologies described in such publications that might be used in connection with Use of Dermal Filler Composition for Treating the present invention . Wrinkles What is claimed is : 5 1. A dermal filler composition comprising: This example illustrates the use of compositions of the a crosslinked hyaluronic acid -based polymer , and invention as a dermal filler for reducing the appearance of a glucosylated derivative of caffeic acid . nasolabial folds . 2. The composition of claim 1, wherein the composition A 62 -year -old woman presents with deep and pronounced is substantially stable during sterilization with a heat treat nasolabial folds . Pre - operative evaluation of the person 10 ment above 100° C. includes routine history and physical examination in addi 3. The composition of claim 1 , wherein free radical tion to thorough informed consent disclosing all relevant stability of the composition is significantly increased by the risks and benefits of the procedure. The physician evaluating addition of the glucosylated derivative of caffeic acid , with the individual determines that she is a candidate for a dermal a Tz, increase of at least about 25 % . filler treatment using the a dermal filler comprising a 15 4. The composition of claim 1 , wherein free radical hyaluronic acid and Epigallocatechin Gallatyl Glucoside, at stability of the composition is significantly increased by the a concentration of 0.5 % , and a concentration of about 15 addition of the glucosylated derivative of caffeic acid , with mg/mL . The composition is administered subcutaneously , a T3, increase of at least about 50 % . using standard techniques , in an amount of about 1.5 ml 5. The composition of claim 1 , wherein free radical under each nasolabial fold ofthe patient's face, through a 27 20 additionstability of the the compositionglucosylated isderivative significantly of caffeic increased acid by , with the gauge needle . The individual is then monitored for approxi a T30 increase of at least about 100 % . mately 14 days . The physician evaluates the facial regions 6. The composition of claim 1 , wherein the composition and determines that the treatment was successful. Both the is injectable. woman and her physician are satisfied with the results of the 7. The composition of claim 1, wherein the crosslinked procedure because the nasolabial folds are less pronounced 25 hyaluronic acid -based polymer is present at a concentration and the patient feels that she looks younger. of about 5 mg/ g to about 40 mg/ g . Although aspects of the present specification have been 8. The composition of claim 1 , wherein the crosslinked described with reference to the various embodiments , one hyaluronic acid -based polymer comprises both high molecu examplesskilled in disclosedthe art will are readilyonly illustrative appreciate of thatthe principlesthe specific of 30 lar weight hyaluronan and low molecular weight hyaluro the subject matter disclosed herein . Therefore , it should be nan . understood that the disclosed subject matter is in no way 9. The composition of claim 8 , wherein the high molecu limited to a particular methodology, protocol, and /or lar weight hyaluronan has a molecular weight greater than reagent, etc., described herein . As such , those skilled in the 2,000,000 Da and wherein the low molecular weight art could make numerous and various modifications or 35 hyaluronan has a molecular weight of less than 1,000,000 changes to or alternative configurations of the disclosed Da . subject matter can be made in accordance with the teachings 10. The composition of claim 1 , wherein the glucosylated herein without departing from the spirit of the present derivative of caffeic acid is present at a concentration of specification . Accordingly , the present invention is not lim about 0.01 % (w /w ) to about 5 % (w / w ). ited to that precisely as shown and described . 40 11. The composition of claim 1 , wherein the composition Groupings of alternative elements or embodiments of the is stable after sterilization with a heat treatment at or about invention disclosed herein are not to be construed as limi 100 ° C. tations . Each group member may be referred to and claimed 12. A method of reducing the appearance of wrinkles in individually or in any combination with other members of skin of a patient , the method comprising administering by the group or other elements found herein . It is anticipated 45 injection , the composition of claim 1 into the skin of the that one or more members of a group may be included in , or patient. deleted from , a group for reasons of convenience and / or 13. A cohesive injectable dermal filler composition com patentability . Unless otherwise indicated , all numbers prising : expressing quantities of ingredients, properties such as a crosslinked hyaluronic acid -based polymer , and a glu molecular weight, reaction conditions, and so forth used in 50 cosylated derivative of caffeic acid , wherein free radi the specification and claims are to be understood as being cal stability of the composition is significantly modified in all instances by the term “ about. ” As used increased by the addition of the glucosylated derivative herein , the term “ about” means that the item , parameter or of caffeic acid , with a T3, increase of at least about term so qualified encompasses a range of plus or minus ten 25 % , and wherein the composition is stable after ster percent above and below the value of the stated item , 55 ilization with a heat treatment at or above 100 ° C. parameter or term . 14. A method of reducing the appearance of wrinkles in All patents , patent publications, and other publications skin of a patient, the method comprising administering by referenced and identified in the present specification are injection , the composition of claim 13 into the skin of the individually and expressly incorporated herein by reference patient. in their entirety for the purpose of describing and disclosing,